Modified Cell Expressing Therapeutic Agent and Uses thereof

ABSTRACT

Compositions and methods for enhancing T cell response which increases the efficacy of CAR T cell therapy for treating cancer are described. Embodiments include a modified cell comprising an isolated nucleic acid comprising a first nucleic acid and a second nucleic acid, the first nucleic acid encoding a chimeric antigen receptor (CAR), the second nucleic acid encoding a therapeutic agent comprising at least one of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23. The modified cell expresses and secretes the therapeutic agent.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims is a continuation of co-pending U.S. patentapplication Ser. No. 16/445,965 filed Jun. 19, 2019, entitled “ModifiedCell Expressing Therapeutic Agent and Uses thereof,” which claims thebenefit of U.S. Provisional Application No. 62/848,961 filed May 16,2019, entitled “Modified Cell Expressing Therapeutic Agent and Usesthereof,” U.S. Provisional Application No. 62/846,563 filed May 10,2019, entitled “Modified Cell Expressing Therapeutic Agent and Usesthereof,” U.S. Provisional Application No. 62/828,770 filed Apr. 3,2019, “Chimeric Antigen Receptor Cell Expressing Therapeutic Agent andUses thereof,” U.S. Provisional Application No. 62/795,810 filed Jan.23, 2019, entitled “Chimeric Antigen Receptor Cell ExpressingTherapeutic Agent and Uses thereof,” U.S. Provisional Application No.62/774,595 filed Dec. 3, 2018, entitled “Chimeric Antigen Receptor CellExpressing Therapeutic Agent and Uses thereof,” and U.S. ProvisionalApplication No. 62/769,987 filed Nov. 20, 2018, entitled “ChimericAntigen Receptor Cell Expressing Therapeutic Agent and Uses thereof,”which are hereby incorporated herein by reference in their entirety.

SEQUENCE LISTING INFORMATION

A computer readable textfile, entitled “Sequence Listing_ST25.bd,”created on or about Aug. 7, 2020 with a file size of about 1.18 MB,contains the sequence listing for this application and is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions and methods related tochimeric antigen receptor cell secreting therapeutic agent, and usesthereof in the treatment of diseases, including cancer.

BACKGROUND

Cancer involves abnormal cell growth with the potential to invade orspread to other parts of the body. In humans, there are more than onehundred types of cancer. One example is breast cancer occurring in theepithelial tissue of the breast. Since breast cancer cells lose thecharacteristics of normal cells, the connection between breast cancercells is lost. Once cancer cells are exfoliated, they spread over theentire body via the blood and/or lymph systems and therefore becomelife-threatening. Currently, breast cancer has become one of the commonthreats to women's physical and mental health. Although immunotherapy,for example CAR T cell therapy, has been proven to be effective fortreating cancer, there is still a need to improve such immunotherapy sothat it is more effective for certain cancers such as those involvingsolid tumors.

SUMMARY

The present disclosure describes compositions and methods for enhancingT cell response. The present disclosure also describes cells comprisingan isolated nucleic acid comprising a nucleic acid and an additionalnucleic acid, the nucleic acid encoding a chimeric antigen receptor(CAR), the additional nucleic acid encoding a therapeutic agentcomprising at least one of IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-17, andIL-23. The cells express and secrete the therapeutic agent.

This Summary is not intended to identify key features or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is described with reference to the accompanyingfigures. The use of the same reference numbers in different figuresindicates similar or identical items.

FIG. 1 is a schematic diagram of an exemplary fusion protein includingGS linkers as set forth in SEQ ID NO: 470 and SEQ ID NO: 124.

FIG. 2 is a schematic diagram of another exemplary fusion proteinincluding GS linkers as set forth in SEQ ID NO: 470 and SEQ ID NO: 124.

FIG. 3 is a schematic diagram of yet another exemplary fusion protein.

FIG. 4 is a schematic diagram of yet another exemplary fusion proteinincluding GS linkers as set forth in SEQ ID NO: 470 and SEQ ID NO: 124.

FIG. 5 is a schematic diagram of an exemplary CAR molecule and a fusionprotein.

FIG. 6 is a schematic diagram of an exemplary CAR molecule and a proteinexpressed by a modified cell.

FIG. 7 is a schematic diagram of another exemplary CAR molecule and oneor more proteins expressed by a modified cell.

FIG. 8 is a schematic diagram of yet another exemplary CAR molecule andone or more proteins expressed by a modified cell.

FIG. 9 is a schematic diagram of yet another exemplary CAR molecule andone or more proteins expressed by a modified cell.

FIG. 10 is a schematic diagram of yet another exemplary CAR molecule andone or more proteins expressed by a modified cell.

FIG. 11 shows PET/CT images showing tumor changes before and after CARTcell infusion.

FIGS. 12, 13, 14, and 15 show changes of cytokine release and otherparameters in response to CAR T cell infusion.

FIG. 16 shows various parameters of the patient in response to CART cellinfusion in a patient.

FIG. 17 shows various parameters of the patient in response to CART cellinfusion in another patient.

FIG. 18 includes various constructs of CAR and therapeutic agents thatmay be expressed by T cells.

FIG. 19 shows flow cytometry assay results of T cells expressing variousproteins shown in FIG. 18.

FIG. 20 shows IL6 release in response to CD3/CD28 Dynabeads activation.

FIG. 21 shows IL6 release in response to co-culturing with Nalm6 cells.

FIG. 22 shows IFNγ (i.e., IFNg) release in response to CD3/CD28Dynabeads activation.

FIG. 23 shows IFNγ release in response to co-culturing with Nalm6 cells.

FIG. 24 shows toxicity assay with respect to CAR T cells.

FIGS. 25 and 26 show other IFNγ release in response to co-culturing withNalm6 cells.

FIGS. 27 and 28 show IL12 and IFNγ release in response to CD3/CD28Dynabeads activation.

FIGS. 29 and 30 show IL6 and IFNγ release in response to CD3/CD28Dynabeads activation.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any method andmaterial similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, preferred methods andmaterials are described. For the purposes of the present disclosure, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

The term “activation,” as used herein, refers to the state of a cellthat has been sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody” is used in the broadest sense and refers tomonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multi-specific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity or function. The antibodies in the presentdisclosure may exist in a variety of forms including, for example,polyclonal antibodies; monoclonal antibodies; Fv, Fab, Fab′, and F(ab)₂and fragments; as well as single chain antibodies and humanizedantibodies (Harlow et al., 1999, In: Using Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989,In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houstonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,1988, Science 242:423-426).

The term “antibody fragments” refers to a portion of a full-lengthantibody, for example, the antigen binding or variable region of theantibody. Other examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multi-specific antibodies formed from antibodyfragments.

The term “Fv” refers to the minimum antibody fragment which contains acomplete antigen-recognition and -binding site. This fragment consistsof a dimer of one heavy- and one light-chain variable region domain intight, non-covalent association. From the folding of these two domainsemanates six hypervariable loops (3 loops each from the H and L chain)that contribute amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv including only three complementaritydetermining regions (CDRs) specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site (the dimer).

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. An “antibody light chain,” asused herein, refers to the smaller of the two types of polypeptidechains present in all antibody molecules in their naturally occurringconformations. K and A light chains refer to the two major antibodylight chain isotypes.

The term “synthetic antibody” refers to an antibody which is generatedusing recombinant DNA technology, such as, for example, an antibodyexpressed by a bacteriophage. The term also includes an antibody whichhas been generated by the synthesis of a DNA molecule encoding theantibody and the expression of the DNA molecule to obtain the antibodyor to obtain an amino acid encoding the antibody. The synthetic DNA isobtained using technology that is available and well known in the art.

The term “antigen” refers to a molecule that provokes an immuneresponse, which may involve either antibody production, or theactivation of specific immunologically-competent cells, or both.Antigens include any macromolecule, including all proteins or peptides,or molecules derived from recombinant or genomic DNA. For example, DNAincluding a nucleotide sequence or a partial nucleotide sequenceencoding a protein or peptide that elicits an immune response, andtherefore, encodes an “antigen” as the term is used herein. An antigenneed not be encoded solely by a full-length nucleotide sequence of agene. An antigen can be generated, synthesized or derived from abiological sample including a tissue sample, a tumor sample, a cell, ora biological fluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect associated with a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, decreasein tumor cell proliferation, decrease in tumor cell survival, anincrease in life expectancy of a subject having tumor cells, oramelioration of various physiological symptoms associated with thecancerous condition. An “anti-tumor effect” can also be manifested bythe ability of the peptides, polynucleotides, cells, and antibodies inthe prevention of the occurrence of tumor in the first place.

The term “auto-antigen” refers to an endogenous antigen mistakenlyrecognized by the immune system as being foreign. Auto-antigens includecellular proteins, phosphoproteins, cellular surface proteins, cellularlipids, nucleic acids, glycoproteins, including cell surface receptors.

The term “autologous” is used to describe a material derived from asubject which is subsequently re-introduced into the same subject.

The term “allogeneic” is used to describe a graft derived from adifferent subject of the same species. As an example, a donor subjectmay be a related or unrelated or recipient subject, but the donorsubject has immune system markers which are similar to the recipientsubject.

The term “xenogeneic” is used to describe a graft derived from a subjectof a different species. As an example, the donor subject is from adifferent species than a recipient subject, and the donor subject andthe recipient subject can be genetically and immunologicallyincompatible.

The term “cancer” is used to refer to a disease characterized by therapid and uncontrolled growth of aberrant cells. Cancer cells can spreadlocally or through the bloodstream and lymphatic system to other partsof the body. Examples of various cancers include breast cancer, prostatecancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, and the like.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “includes” and “including” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

The phrase “consisting of” is meant to include, and is limited to,whatever follows the phrase “consisting of.” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory and that no other elements may be present.

The phrase “consisting essentially of” is meant to include any elementlisted after the phrase and can include other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides(i.e., a sequence of nucleotides) related by the base-pairing rules. Forexample, the sequence “A-G-T,” is complementary to the sequence “T-C-A.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules, or theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands.

The term “corresponds to” or “corresponding to” refers to (a) apolynucleotide having a nucleotide sequence that is substantiallyidentical or complementary to all or a portion of a referencepolynucleotide sequence or encoding an amino acid sequence identical toan amino acid sequence in a peptide or protein; or (b) a peptide orpolypeptide having an amino acid sequence that is substantiallyidentical to a sequence of amino acids in a reference peptide orprotein.

The term “co-stimulatory ligand,” refers to a molecule on an antigenpresenting cell (e.g., an APC, dendritic cell, B cell, and the like)that specifically binds a cognate co-stimulatory molecule on a T cell,thereby providing a signal which, in addition to the primary signalprovided by, for instance, binding of a TCR/CD3 complex with an MHCmolecule loaded with peptide, mediates a T cell response, including atleast one of proliferation, activation, differentiation, and othercellular responses. A co-stimulatory ligand can include B7-1 (CD80),B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatoryligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40,CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6,ILT3, ILT4, HVEM, a ligand for CD7, an agonist or antibody that bindsthe Toll ligand receptor and a ligand that specifically binds withB7-H3. A co-stimulatory ligand also includes, inter alia, an agonist oran antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds CD83.

The term “co-stimulatory molecule” refers to the cognate binding partneron a T cell that specifically binds with a co-stimulatory ligand,thereby mediating a co-stimulatory response by the T cell, such asproliferation. Co-stimulatory molecules include an MHC class I molecule,BTLA, and a Toll-like receptor.

The term “co-stimulatory signal” refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules. The terms “disease” and “condition” may be usedinterchangeably or may be different in that the particular malady orcondition may not have a known causative agent (so that etiology has notyet been worked out), and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians. Theterm “disease” is a state of health of a subject wherein the subjectcannot maintain homeostasis, and wherein if the disease is notameliorated then the subject's health continues to deteriorate. Incontrast, a “disorder” in a subject is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

The term “effective” refers to adequate to accomplish a desired,expected, or intended result. For example, an “effective amount” in thecontext of treatment may be an amount of a compound sufficient toproduce a therapeutic or prophylactic benefit.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as a template for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene encodes a protein if transcription and translation of mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. Both the coding strand, the nucleotide sequence ofwhich is identical to the mRNA sequence (except that a “T” is replacedby a “U”) and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

The term “exogenous” refers to a molecule that does not naturally occurin a wild-type cell or organism but is typically introduced into thecell by molecular biological techniques. Examples of exogenouspolynucleotides include vectors, plasmids, and/or man-made nucleic acidconstructs encoding the desired protein. With regard to polynucleotidesand proteins, the term “endogenous” or “native” refers tonaturally-occurring polynucleotide or amino acid sequences that may befound in a given wild-type cell or organism. Also, a particularpolynucleotide sequence that is isolated from a first organism andtransferred to a second organism by molecular biological techniques istypically considered an “exogenous” polynucleotide or amino acidsequence with respect to the second organism. In specific embodiments,polynucleotide sequences can be “introduced” by molecular biologicaltechniques into a microorganism that already contains such apolynucleotide sequence, for instance, to create one or more additionalcopies of an otherwise naturally-occurring polynucleotide sequence, andthereby facilitate overexpression of the encoded polypeptide.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by its promoter.

The term “expression vector” refers to a vector including a recombinantpolynucleotide including expression control (regulatory) sequencesoperably linked to a nucleotide sequence to be expressed. An expressionvector includes sufficient cis-acting elements for expression; otherelements for expression can be supplied by the host cell or in an invitro expression system. Expression vectors include all those known inthe art, such as cosmids, plasmids (e.g., naked or contained inliposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses,and adeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “homologous” refers to sequence similarity or sequence identitybetween two polypeptides or between two polynucleotides when a positionin both of the two compared sequences is occupied by the same base oramino acid monomer subunit, e.g., if a position in each of two DNAmolecules is occupied by adenine, then the molecules are homologous atthat position. The percent of homology between two sequences is afunction of the number of matching or homologous positions shared by thetwo sequences divided by the number of positions compared ×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous, then the two sequences are 60% homologous. By way ofexample, the DNA sequences ATTGCC and TATGGC share 50% homology. Acomparison is made when two sequences are aligned to give maximumhomology.

The term “immunoglobulin” or “Ig,” refers to a class of proteins, whichfunction as antibodies. The five members included in this class ofproteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibodythat is present in body secretions, such as saliva, tears, breast milk,gastrointestinal secretions and mucus secretions of the respiratory andgenitourinary tracts. IgG is the most common circulating antibody. IgMis the main immunoglobulin produced in the primary immune response inmost subjects. It is the most efficient immunoglobulin in agglutination,complement fixation, and other antibody responses, and is important indefense against bacteria and viruses. IgD is the immunoglobulin that hasno known antibody function but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingthe release of mediators from mast cells and basophils upon exposure tothe allergen.

The term “isolated” refers to a material that is substantially oressentially free from components that normally accompany it in itsnative state. The material can be a cell or a macromolecule such as aprotein or nucleic acid. For example, an “isolated polynucleotide,” asused herein, refers to a polynucleotide, which has been purified fromthe sequences which flank it in a naturally-occurring state, e.g., a DNAfragment which has been removed from the sequences that are normallyadjacent to the fragment. Alternatively, an “isolated peptide” or an“isolated polypeptide” and the like, as used herein, refer to in vitroisolation and/or purification of a peptide or polypeptide molecule fromits natural cellular environment, and from association with othercomponents of the cell.

The term “substantially purified” refers to a material that issubstantially free from components that are normally associated with itin its native state. For example, a substantially purified cell refersto a cell that has been separated from other cell types with which it isnormally associated in its naturally occurring or native state. In someinstances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to a cell that has been separated from the cells with which theyare naturally associated in their natural state. In embodiments, thecells are cultured in vitro. In embodiments, the cells are not culturedin vitro.

In the context of the present disclosure, the following abbreviationsfor the commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. Moreover, the use oflentiviruses enables integration of the genetic information into thehost chromosome resulting in stably transduced genetic information. HIV,SIV, and FIV are all examples of lentiviruses. Vectors derived fromlentiviruses offer the means to achieve significant levels of genetransfer in vivo.

The term “modulating,” refers to mediating a detectable increase ordecrease in the level of a response in a subject compared with the levelof a response in the subject in the absence of a treatment or compound,and/or compared with the level of a response in an otherwise identicalbut untreated subject. The term encompasses perturbing and/or affectinga native signal or response thereby mediating a beneficial therapeuticresponse in a subject, preferably, a human.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation.

The term “under transcriptional control” refers to a promoter beingoperably linked to and in the correct location and orientation inrelation to a polynucleotide to control (regulate) the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

The term “overexpressed” tumor antigen or “overexpression” of the tumorantigen is intended to indicate an abnormal level of expression of thetumor antigen in a cell from a disease area such as a solid tumor withina specific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

Cancers that may be treated include tumors that are not vascularized, ornot yet substantially vascularized, as well as vascularized tumors. Thecancers may include non-solid tumors (such as hematological tumors, forexample, leukemias and lymphomas) or may include solid tumors. Types ofcancers to be treated with the CARs of the disclosure include, but arenot limited to, carcinoma, blastoma, and sarcoma, and certain leukemiaor lymphoid malignancies, benign and malignant tumors, and malignancies,e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers andpediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples ofhematological (or hematogenous) cancers include leukemias, includingacute leukemias (such as acute lymphocytic leukemia, acute myelocyticleukemia, acute myelogenous leukemia and myeloblastic, promyelocytic,myelomonocytic, monocytic and erythroleukemia), chronic leukemias (suchas chronic myelocytic (granulocytic) leukemia, chronic myelogenousleukemia, and chronic lymphocytic leukemia), polycythemia vera,lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and highgrade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavychain disease, myelodysplastic syndrome, hairy cell leukemia andmyelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, and brainmetastases).

A solid tumor antigen is an antigen expressed on a solid tumor. Inembodiments, solid tumor antigen is also expressed at low levels onhealthy tissue. Examples of solid tumor antigens and their relateddisease tumors are provided in Table 1.

TABLE 1 Solid Tumor antigen Disease tumor PRLR Breast Cancer CLCA1colorectal Cancer MUC12 colorectal Cancer GUCY2C colorectal Cancer GPR35colorectal Cancer CR1L Gastric Cancer MUC 17 Gastric Cancer TMPRSS11Besophageal Cancer MUC21 esophageal Cancer TMPRSS11E esophageal CancerCD207 bladder Cancer SLC30A8 pancreatic Cancer CFC1 pancreatic CancerSLC12A3 Cervical Cancer SSTR1 Cervical tumor GPR27 Ovary tumor FZD10Ovary tumor TSHR Thyroid Tumor SIGLEC15 Urothelial cancer SLC6A3 Renalcancer KISS1R Renal cancer QRFPR Renal cancer: GPR119 Pancreatic cancerCLDN6 Endometrial cancer/Urothelial cancer UPK2 Urothelial cancer(including bladder cancer) ADAM12 Breast cancer, pancreatic cancer andthe like SLC45A3 Prostate cancer ACPP Prostate cancer MUC21 Esophagealcancer MUC16 Ovarian cancer MS4A12 Colorectal cancer ALPP Endometrialcancer CEA Colorectal carcinoma EphA2 Glioma FAP Mesotelioma GPC3 Lungsquamous cell carcinoma IL13-Rα2 Glioma Mesothelin Metastatic cancerPSMA Prostate cancer ROR1 Breast lung carcinoma VEGFR-II Metastaticcancer GD2 Neuroblastoma FR-α Ovarian carcinoma ErbB2 Carcinomasb EpCAMCarcinomasa EGFRvIII Glioma-Glioblastoma EGFR Glioma-NSCL cancer tMUC 1Cholangiocarcinoma, Pancreatic cancer, Breast Cancer B7-H3 Ewing sarcoma(bone tumor), rhabdomyosarcoma, nephroblastoma, neuroblastoma andmedulloblastoma (brain tumor)

The term “parenteral administration” of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.),intrasternal injection, or infusion techniques.

The terms “patient,” “subject,” and “individual,” and the like are usedinterchangeably herein and refer to any human, or animal, amenable tothe methods described herein. In certain non-limiting embodiments, thepatient, subject, or individual is a human or animal. In embodiments,the term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, and animals such as dogs, cats, mice, rats, andtransgenic species thereof.

A subject in need of treatment or in need thereof includes a subjecthaving a disease, condition, or disorder that needs to be treated. Asubject in need thereof also includes a subject that needs treatment forprevention of a disease, condition, or disorder. In embodiments, thedisease, condition, or disorder is cancer.

The term “polynucleotide” or “nucleic acid” refers to mRNA, RNA, cRNA,rRNA, cDNA or DNA. The term typically refers to a polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes all forms of nucleic acids including single anddouble-stranded forms of nucleic acids.

The terms “polynucleotide variant” and “variant” and the like refer topolynucleotides displaying substantial sequence identity with areference polynucleotide sequence or polynucleotides that hybridize witha reference sequence under stringent conditions that are definedhereinafter. These terms also encompass polynucleotides that aredistinguished from a reference polynucleotide by the addition, deletionor substitution of at least one nucleotide. Accordingly, the terms“polynucleotide variant” and “variant” include polynucleotides in whichone or more nucleotides have been added or deleted or replaced withdifferent nucleotides. In this regard, it is well understood in the artthat certain alterations inclusive of mutations, additions, deletions,and substitutions can be made to a reference polynucleotide whereby thealtered polynucleotide retains the biological function or activity ofthe reference polynucleotide or has increased activity in relation tothe reference polynucleotide (i.e., optimized). Polynucleotide variantsinclude, for example, polynucleotides having at least 50% (and at least51% to at least 99% and all integer percentages in between, e.g., 90%,95%, or 98%) sequence identity with a reference polynucleotide sequencedescribed herein. The terms “polynucleotide variant” and “variant” alsoinclude naturally-occurring allelic variants and orthologs.

The terms “polypeptide,” “polypeptide fragment,” “peptide,” and“protein” are used interchangeably herein to refer to a polymer of aminoacid residues and to variants and synthetic analogues of the same. Thus,these terms apply to amino acid polymers in which one or more amino acidresidues are synthetic non-naturally occurring amino acids, such as achemical analogue of a corresponding naturally occurring amino acid, aswell as to naturally-occurring amino acid polymers. In certain aspects,polypeptides may include enzymatic polypeptides, or “enzymes,” whichtypically catalyze (i.e., increase the rate of) various chemicalreactions.

The term “polypeptide variant” refers to polypeptides that aredistinguished from a reference polypeptide sequence by the addition,deletion, or substitution of at least one amino acid residue. In certainembodiments, a polypeptide variant is distinguished from a referencepolypeptide by one or more substitutions, which may be conservative ornon-conservative. In certain embodiments, the polypeptide variantcomprises conservative substitutions and, in this regard, it is wellunderstood in the art that some amino acids may be changed to otherswith broadly similar properties without changing the nature of theactivity of the polypeptide. Polypeptide variants also encompasspolypeptides in which one or more amino acids have been added or deletedor replaced with different amino acid residues.

The term “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence. Theterm “expression control (regulatory) sequences” refers to DNA sequencesnecessary for the expression of an operably linked coding sequence in aparticular host organism. The control (regulatory) sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

The term “bind,” “binds,” or “interacts with” refers to a moleculerecognizing and adhering to a second molecule in a sample or organismbut does not substantially recognize or adhere to other structurallyunrelated molecules in the sample. The term “specifically binds,” asused herein with respect to an antibody, refers to an antibody whichrecognizes a specific antigen, but does not substantially recognize orbind other molecules in a sample. For example, an antibody thatspecifically binds an antigen from one species may also bind thatantigen from one or more species. But, such cross-species reactivitydoes not itself alter the classification of an antibody as specific. Inanother example, an antibody that specifically binds an antigen may alsobind different allelic forms of the antigen. However, such crossreactivity does not itself alter the classification of an antibody asspecific. In some instances, the terms “specific binding” or“specifically binding,” can be used in reference to the interaction ofan antibody, a protein, or a peptide with a second chemical species, tomean that the interaction is dependent upon the presence of a particularstructure (e.g., an antigenic determinant or epitope) on the chemicalspecies; for example, an antibody recognizes and binds a specificprotein structure rather than to any protein. If an antibody is specificfor epitope “A,” the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

By “statistically significant,” it is meant that the result was unlikelyto have occurred by chance. Statistical significance can be determinedby any method known in the art. Commonly used measures of significanceinclude the p-value, which is the frequency or probability with whichthe observed event would occur if the null hypothesis were true. If theobtained p-value is smaller than the significance level, then the nullhypothesis is rejected. In simple cases, the significance level isdefined at a p-value of 0.5 or less. A “decreased” or “reduced” or“lesser” amount is typically a “statistically significant” or aphysiologically significant amount, and may include a decrease that isabout 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4,4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100,500, 1000 times) (including all integers and decimal points in betweenand above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) an amount or leveldescribed herein.

The term “stimulation,” refers to a primary response induced by bindingof a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognateligand thereby mediating a signal transduction event, such as signaltransduction via the TCR/CD3 complex. Stimulation can mediate alteredexpression of certain molecules, such as downregulation of TGF-β, and/orreorganization of cytoskeletal structures.

The term “stimulatory molecule” refers to a molecule on a T cell thatspecifically binds a cognate stimulatory ligand present on an antigenpresenting cell. For example, a functional signaling domain derived froma stimulatory molecule is the zeta chain associated with the T cellreceptor complex. The stimulatory molecule includes a domain responsiblefor signal transduction.

The term “stimulatory ligand” refers to a ligand that when present on anantigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, andthe like.) can specifically bind with a cognate binding partner(referred to herein as a “stimulatory molecule”) on a cell, for examplea T cell, thereby mediating a primary response by the T cell, includingactivation, initiation of an immune response, proliferation, and similarprocesses. Stimulatory ligands are well-known in the art and encompass,inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2antibody.

The term “therapeutic” refers to a treatment and/or prophylaxis. Atherapeutic effect is obtained by suppression, remission, or eradicationof a disease state or alleviating the symptoms of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or another clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent the development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

The term “treat a disease” refers to the reduction of the frequency orseverity of at least one sign or symptom of a disease or disorderexperienced by a subject.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which an exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed, or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “vector” refers to a polynucleotide that comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding linear polynucleotides, polynucleotides associated with ionicor amphiphilic compounds, plasmids, and viruses. Thus, the term “vector”includes an autonomously replicating plasmid or a virus. The term alsoincludes non-plasmid and non-viral compounds which facilitate thetransfer of nucleic acid into cells, such as, for example, polylysinecompounds, liposomes, and the like. Examples of viral vectors includeadenoviral vectors, adeno-associated virus vectors, retroviral vectors,and others. For example, lentiviruses are complex retroviruses, which,in addition to the common retroviral genes gag, pol, and env, containother genes with regulatory or structural function. Lentiviral vectorsare well known in the art. Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2, and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu, and nef are deleted making the vector biologically safe.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

A “chimeric antigen receptor” (CAR) molecule is a recombinantpolypeptide including at least an extracellular domain, a transmembranedomain and a cytoplasmic domain or intracellular domain. In embodiments,the domains of the CAR are on the same polypeptide chain, for example achimeric fusion protein. In embodiments, the domains are on differentpolypeptide chains, for example the domains are not contiguous.

The extracellular domain of a CAR molecule includes an antigen bindingdomain. In embodiments, the antigen binding domain binds an antigen, forexample, a cell surface molecule or marker, on the surface of a B cell.In embodiments, the cell surface molecule of a B cell includes CD19,CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b,CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cellsurface molecule of the B cell is CD19, CD20, CD22, or BCMA. Inparticular embodiments, the cell surface molecule of the B cell is CD19.

In embodiments, the antigen binding domain binds an antigen, on thesurface of a tumor for example a tumor antigen or tumor marker. Tumorantigens are proteins that are produced by tumor cells that elicit animmune response, particularly T cell mediated immune responses. Tumorantigens are well known in the art and include, for example, tumorassociated MUC1 (tMUC1), a glioma-associated antigen, carcinoembryonicantigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP),lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerasereverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP,NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, surviving, telomerase,prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophilelastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor and mesothelin. For example, when the tumor antigen is CD19,and the CAR thereof can be referred to as CD19CAR.

In embodiments, the extracellular antigen binding domain of a CARincludes at least one scFv or at least a single domain antibody. As anexample, there can be two scFvs on a CAR. The scFv includes a lightchain variable (VL) region and a heavy chain variable (VH) region of atarget antigen-specific monoclonal antibody joined by a flexible linker.Single chain variable region fragments can be made by linking lightand/or heavy chain variable regions by using a short linking peptide(Bird et al., Science 242:423-426, 1988). An example of a linkingpeptide is the GS linker having the amino acid sequence (GGGGS)₃ SEQ IDNO: 124, which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used (Bird etal., 1988, supra). In general, linkers can be short, flexiblepolypeptides and preferably comprised of about 20 or fewer amino acidresidues. The single chain variants can be produced either recombinantlyor synthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

In embodiments, the CAR molecules described herein comprises one or moreCDRs for binding an antigen of interest, for example, one or more CDRsof CD19 or tMUC1.

The cytoplasmic domain of the CAR molecules described herein includesone or more co-stimulatory domains and one or more signaling domains.The co-stimulatory and signaling domains function to transmit the signaland activate molecules, such as T cells, in response to antigen binding.The one or more co-stimulatory domains are derived from stimulatorymolecules and/or co-stimulatory molecules, and the signaling domain isderived from a primary signaling domain, such as the CD3 zeta domain. Inembodiments, the signaling domain further includes one or morefunctional signaling domains derived from a co-stimulatory molecule. Inembodiments, the co-stimulatory molecules are cell surface molecules(other than antigens receptors or their ligands) that are required foractivating a cellular response to an antigen.

In embodiments, the co-stimulatory domain includes the intracellulardomain of CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds CD83, or any combination thereof. Inembodiments, the signaling domain includes a CD3 zeta domain derivedfrom a T cell receptor.

In embodiments, the cytoplasmic domain of the CAR only includes one ormore stimulatory domains and no signaling domain.

The CAR molecules also include a transmembrane domain. The incorporationof a transmembrane domain in the CAR molecules stabilizes the molecule.In embodiments, the transmembrane domain of the CAR molecules is thetransmembrane domain of a CD28 or 4-1BB molecule.

Between the extracellular domain and the transmembrane domain of theCAR, there may be incorporated a spacer domain. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the extracellular domain or,the cytoplasmic domain on the polypeptide chain. A spacer domain mayinclude up to 300 amino acids, preferably 10 to 100 amino acids, andmost preferably 25 to 50 amino acids.

CAR Molecule(s) and Therapeutic Agent(s)

The present disclosure describes isolated nucleic acids including a(first) nucleic acid encoding a CAR and an additional (second) nucleicacid encoding one or more therapeutic agents. In embodiments, the firstnucleic acid and the second nucleic acid are on separate isolatednucleic acids. In embodiments, the first and second nucleic acid are onthe same isolated nucleic acid. In embodiments, the one or moretherapeutic agents is IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-17, IL-23, or acombination thereof. In embodiments, the therapeutic agent is Eomes,TRAF6, IL12, IL2, IL18, IL23, AQP9, Runx3, AMPK, BCL-2, or a combinationthereof.

The present disclosure also describes isolated nucleic acids including afirst nucleic acid encoding a first CAR and a second nucleic acidencoding the one or more therapeutic agents. Moreover, the presentdisclosure describes isolated nucleic acids including a third nucleicacid encoding a second (or additional) CAR. In embodiments, a separateisolated nucleic acid includes a nucleic acid encoding the second CAR.In embodiments, the first nucleic acid and the second nucleic acid areon separate isolated nucleic acids.

In embodiments, that first CAR includes an antigen binding domain thatbinds a solid tumor, and the second (or additional) CAR includes anantigen binding domain that binds a white blood cell (WBC).

The present disclosure also describes vectors including the isolatednucleic acids described above. In embodiments, a single vector containsthe isolated nucleic acid encoding the first CAR, the therapeutic agent,and the second CAR or TCR. In embodiments, a first vector contains thefirst nucleic acid encoding a first CAR and a nucleic acid encoding oneor more therapeutic agents, and a second vector contains the nucleicacid encoding the second CAR or TCR. In embodiments, the vectorcomprises an isolated nucleic acid encoding a bispecific CAR includingat least two antigen binding domains and one or more therapeutic agents.

In embodiments, the present disclosure describes an isolated nucleicacid sequence comprising a nucleic acid sequence and an additionalnucleic acid sequence, the nucleic acid sequence encoding a chimericantigen receptor (CAR), the additional nucleic acid sequence encoding atherapeutic agent that is or comprises at least one of IL-2, IL-6, IL-7,IL-15, IL-17, and IL-23. In embodiments, the therapeutic agent is orcomprises Eomes, TRAF6, IL12, IL2, IL18, IL23, AQP9, Runx3, AMPK, orBCL-2.

In embodiments, the present disclosure describes a pharmaceuticalcomposition for treating a subject having a tumor using modified Tcells, wherein the pharmaceutical composition comprises modified T cellscomprising a first nucleic acid sequence encoding a chimeric antigenreceptor (CAR) and a second nucleic acid encoding a therapeutic agentcomprising IL-6, IFN-γ, or a combination thereof. In embodiments, thetumor is a solid tumor. In embodiments, the tumor is a liquid tumor(e.g., NHL). Embodiments relate to a method of causing or inducing Tcell response in a subject in need thereof, the method comprisingadministering an effective amount of the pharmaceutical composition tothe subject. The method described herein is effective in treating asubject diagnosed with cancer. In embodiments, the subject is diagnosedwith a solid tumor.

Embodiments relate to certain cytokines, for example IL-6 and IFNγ, areselected to be expressed or overexpressed in T cells, which are used totreat tumors (e.g., solid and/or liquid tumors). These cytokines atleast do not directly or indirectly weaken the killing function,capability of inhibiting tumor cells, and/or has severe side effects onT cell therapy. For example, these selected cytokines are capable ofenhancing T cell response. Interestingly, IL-6 was considered as acytokine that reduces or at least has a negative impact on T celltherapy since it is the major contributor to Cytokine Release Syndrome(CRS). However, the Examples provided herein show that the increase ofIL-6 is consistent with the efficacy in treating Relapsed/Refractory(R/R) Acute Lymphoid Leukemia (ALL) using CAR T cell therapy.Surprisingly, the Examples provided herein show infusion of CAR T cellsexpressing and secreting IL-6 do not cause severe CRS for treating solidtumors. Not all cytokine can be expressed and secreted by T cellswithout sacrificing their function to kill tumor cells and/or inhibittumor growth. The tumor-promoting effects of certain cytokines have beenreported. For example, IL-10 production by TAMs can blunt anti-tumorresponses by inhibiting the functions of APCs and subsequently block Tcell effector functions such as cytotoxicity (Mannino, M. H., Zhu, Z.,Xiao, H., Bai, Q., Wakefield, M. R., and Fang, Y. (2015). Studies inmouse tumor models have shown that IL-10 can suppress tumor-infiltratingDC maturation and their production of IL-12 to stimulate Th1 cells,unless IL-10 signaling is simultaneously blocked (Vicari, A. P.,Chiodoni, C., Vaure, C., Ai{umlaut over ( )}t-Yahia, S., Dercamp, C.,Matsos, F., Reynard, O., Taverne, C., Merle, P., Colombo, M. P., et al.(2002). Reversal of tumor-induced dendritic cell paralysis by CpGimmunostimulatory oligonucleotide and anti-interleukin 10 receptorantibody. J. Exp. Med. 196, 541-549.). As another example, studies haveshown that TGF-β can be a potent inhibitor of T cell proliferation(Kehrl J H, Wakefield L M, Roberts A B, Jakowlew S, Alvarez-Mon M, etal. 1986. Production of transforming growth factor β (TGF-β) by human Tlymphocytes and its potential role in the regulation of T cell growth.J. Exp. Med. 163:1037-50). Several mechanisms drive TGF-β-mediatedinhibition of T cell proliferation, including suppression of IL-2production, downregulation of c-myc, and upregulation ofcyclin-dependent kinase inhibitors (Li M O, Wan Y Y, Sanjabi S,Robertson A K, Flavell R A. 2006. Transforming growth factor-βregulation of immune responses. Annu. Rev. Immunol. 24:99-146). In somecontexts, TGF-β also plays an important role in promoting cell death tolimit T cell expansion after activation (Mark A. Travis and DeanSheppard, TGF-8 Activation and Function in Immunity, Annu. Rev. Immunol.2014. 32:51-82). Chemokines are a large family of cytokines that directnormal leukocyte migration. They also have been implicated in leukocytedevelopment and in the pathogenesis of many diseases. Also, somechemokines' concentration gradients play an important role in intranodalT-cell migration. Overexpression of these chemokines would disrupt Tcells migration, thus weakening CAR T cell therapy for solid tumor.Without proper migration, T cells may not be able to reach tumor cells.For example, it has been reported that overexpression of the chemokineCCL21 disrupts T cell migration (Christopherson K W and Campbell J J,Hromas R A, Transgenic overexpression of the CC chemokine CCL21 disruptsT-cell migration, Blood. 2001 Dec. 15; 98(13):3562-8). In embodiments,cytokines over-expressed or expressed in the modified cells does notinclude at least one of IL-10, TGF-β. and CCL21. Certain cytokines canbe overexpressed or expressed in T cells to enhance CAR T therapytreating tumors. However, some cytokines (e.g., IL-6) cannot beoverexpressed or expressed in T cells to treat blood tumor. It iswell-known that IL-6 is the major factor that contributes to severe CRSin CAR T treatment of blood tumors such as ALL and NHL. However, IL-6can be overexpressed or expressed in T cells for the treatment of solidtumors because there are few studies reporting severe CRS in CAR T celltreatment of solid tumor. In embodiments, expression and secretion ofIL-6 by T cells may be associated with a condition of T cells to avoidCRS and other syndromes related to IL-6. For example, IL-6 may beexpressed and secreted by T cells when the T cells are activated. Inembodiments, expression and secretion of IL-6 by CAR T cells may beregulated by a transcription modulator such as NFAT such that the CAR Tcells may neither express nor secrete IL-6 unless they recognize andbind their antigen.

In embodiments, the modified T cells express and secrete the therapeuticagent. In embodiments, the therapeutic agent comprises IL-6 and IFN-γ.In embodiments, the modified T cell comprises nucleic acid sequencesencoding SEQ ID NOS: 287 and 328. In embodiments, the modified T cellcomprises the nucleic acid sequences comprises SEQ ID NOS: 286 and 469,and a nucleic acid sequence encodes SEQ ID NO: 328.

In embodiments, the CAR comprises an extracellular domain, atransmembrane domain, and an intracellular domain, the extracellulardomain binds an antigen. In embodiments, the CAR binds tMUC 1, PRLR,CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E,CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15,SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP,MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Ra2, Mesothelin,PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, PSCA, or EGFR.In embodiments, the intracellular domain comprises a co-stimulatorydomain that comprises an intracellular domain of a co-stimulatorymolecule selected from the group consisting of CD27, CD28, 4-1BB, OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof. Inembodiments, the antigen is Epidermal growth factor receptor (EGFR),Variant III of the epidermal growth factor receptor (EGFRvIII), Humanepidermal growth factor receptor 2 (HER2), Mesothelin (MSLN),Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen(CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.

In embodiments, the therapeutic agent is present in the modified T cellin a recombinant DNA construct, in an mRNA, or in a viral vector. Inembodiments, the modified T cell comprises a nucleic acid sequencecomprising a promoter comprising a binding site for a transcriptionmodulator that modulates the expression and/or secretion of thetherapeutic agent in the modified cell. In embodiments, thetranscription modulator is or comprises Hif1a, NFAT, FOXP3, or NFkB. Inembodiments, the promoter is responsive to the transcription modulator.In embodiments, the promoter is operably linked to the nucleic acidsequence encoding the therapeutic agent such that the promoter drivesexpression and/or secretion of the therapeutic agent. In embodiments,the promoter comprises at least one of SEQ ID Nos: 323-325.

In embodiments, the CAR and the therapeutic agent are produced in theform of a polyprotein, which is cleaved to generate separate CAR andtherapeutic agent molecules, and there is a cleavable moiety between theCAR and the therapeutic agent, the cleavable moiety comprises a 2Apeptide, the 2A peptide comprises P2A or T2A.

In embodiments, the modified T cell comprises an additional (second)CAR, the CAR binds a solid tumor antigen, and the additional CAR bindsan antigen of a white blood cell. In some embodiments, the solid tumorantigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17,TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1,GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6,UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2,FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2,EpCAM, EGFRvIII, PSCA, or EGFR, and the antigen of the white blood cellis CD19, CD20, CD22, or BCMA. In embodiments, the modified cellcomprises a dominant negative PD-1.

Embodiments relate to an isolated nucleic acid comprising a nucleic acidsequence and an additional nucleic acid sequence, the nucleic acidencoding a chimeric antigen receptor (CAR), the additional nucleic acidencoding a therapeutic agent that comprises at least one of TNFRSFsuperfamily member receptor activation antibodies or membrane-boundforms thereof, TNFRSF superfamily member ligands or the membrane-boundform thereof, chemokines or membrane-bound forms thereof, antibodies tothe chemokines, or antibodies to receptors of the chemokines or themembrane-bound forms thereof, and D28 family's ligands that correspondto the sequences in Table 2-4. For example, TNFRSF superfamily memberreceptor includes tumor necrosis factor receptor 1, Tumor necrosisfactor receptor 2, Lymphotoxin beta receptor, Lymphotoxin beta receptor,CD40, Fas receptor, Decoy receptor 3, CD27, CD30, 4-1BB, Death receptor4, Death receptor 5, Decoy receptor 1, Decoy receptor 2, RANK,Osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, Herpesvirus entrymediator, Nerve growth factor receptor, B-cell maturation antigen,Glucocorticoid-induced TNFR-related, TROY, Death receptor 6, Deathreceptor 3, Ectodysplasin A2 receptor, and the like.

In embodiments, the therapeutic agent includes an antibody reagent(e.g., a single chain antibody (e.g., scFv), a single domain antibody(e.g., a camelid antibody), or a bispecific antibody reagent (e.g., abispecific T cell engager (BITE)). In embodiments, the therapeutic agentincludes a cytokine. Examples of the cytokines include IL-1P, IL-2,IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-1Ra,IL-2R, IFN-γ, IFN-γ, MIP-In, MIP-IP, MCP-1, TNFα, GM-CSF, GCSF, CXCL9,CXCL10, CXCR factors, VEGF, RANTES, EOTAXIN, EGF, HGF, FGF-P, CD40,CD40L, ferritin, and any combination thereof. In embodiments, thecytokines include proinflammatory cytokines such as: IFN-γ, IL-15, IL-4,IL-10, TNFα, IL-8, IL-5, IL-6, GM-CSF, and MIP-Iα. For example, IFN-γhas been approved by FDA to treat patients with malignant osteoporosis(e.g., Journal of Pediatrics 121(1):119-24⋅August 1992).

T present disclosure describes a population of CAR cells comprising thenucleic acid and the additional nucleic acid, wherein the CAR cellscomprise lymphocyte, leukocyte, or PBMC. In embodiments, the CAR and thetherapeutic agent are produced in the form of a polyprotein, which iscleaved to generate separate CAR and therapeutic agent molecules. Inembodiments, the polyprotein comprises a cleavable moiety between theCAR and the therapeutic agent, the cleavable moiety comprising a 2Apeptide, the 2A peptide comprises P2A or T2A. In embodiments, the CARand the therapeutic agent are each constitutively expressed. Inembodiments, the CAR cells comprise: a third nucleic acid sequenceencoding an additional CAR binding to an antigen that is different fromthe CAR, or the additional CAR binding a solid tumor antigen, and theCAR binds an antigen of a white blood cell. In embodiments, thetherapeutic agent or its variants can be produced either recombinantlyor synthetically. For synthetic production of the therapeutic agent, anautomated synthesizer can be used. For recombinant production of thetherapeutic agent, a suitable plasmid containing polynucleotide thatencodes the therapeutic agent can be introduced into a suitable hostcell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thetherapeutic agent of interest can be made by routine manipulations suchas ligation of polynucleotides. The resultant therapeutic agent can beisolated using standard protein purification techniques known in theart.

The present disclosure describes a pharmaceutical composition comprisingthe population of the CAR cells. Embodiments relate to a method ofinducing or enhancing T cell response in a subject in need thereofand/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition to the subject.

The present disclosure describes a modified cell comprising one or moreCARs, wherein the cell is engineered to express and secrete one or moretherapeutic agents comprising at least one of IL-2, IL-6, IL-7, IL-15,IL-17, and IL-23. In embodiments, the cell is engineered to express thetherapeutic agent, which is bound to the membrane of the modified cell.

The present disclosure describes a method of causing or enhancing T cellresponse, treating cancer, or enhancing cancer treatment, the methodcomprising: administrating an effective amount of the composition of Tcells comprising one or more CARs, wherein the cell is engineered toexpress and secrete a therapeutic agent that is or comprises at leastone of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23, and the T cellresponse is enhanced as compared to the administration of T cells thatdo not express or secrete the therapeutic agent.

The present disclosure describes a method of causing or enhancing T cellresponse, treating cancer, or enhancing cancer treatment, the methodcomprising: administering an effective amount of the composition of apopulation of T cells comprising a CAR; and administering an effectiveamount of a therapeutic agent comprising at least one of IL-2, IL-6,IL-7, IL-15, IL-17, and IL-23, wherein the T cell response is enhancedas compared to the administration of CAR T cells without theadministration of therapeutic agent. In embodiments, the administeringthe effective amount of the therapeutic agent comprises intravenousdelivery of an amount of human IL-6 in the range of about 0.5-50 ug perkilogram of body weight. In embodiments, the therapeutic agent is IL-6or IL-7.

In embodiments, the method further comprises monitoring a concentrationof the therapeutic agent in tissue or blood of the subject; andadministering an antagonist of receptors of the therapeutic agent or thetherapeutic agent (e.g., antibodies) if the concentration and/or otherparameters of the subject are not in a desired condition. For example,the parameters may include a level of body temperatures, a level CRS,and a level of neuronal toxicity etc.

In embodiments, the expression and/or secretion of the therapeutic agentmay be is regulated by an inducible expression system. In embodiments,the inducible expression system is a rtTA-TRE system, which increases oractivates the expression of therapeutic agent, or a combination thereof.In embodiments, the inducible expression system is the rtTA-TRE system.For example, Tetracycline-Controlled Transcriptional Activation is amethod of inducible gene expression where transcription is reversiblyturned on or off in the presence of the antibiotic tetracycline or oneof its derivatives (e.g., doxycycline). In embodiments, the expressionand/or secretion of the therapeutic agent may be regulated by aninducible expression system and/or the modified cell comprises a nucleicacid sequence encoding an inducible suicide system. For example, theinducible suicide system is an HSV-TK system or an inducible caspase-9system.

In embodiments, the T cell comprises an additional (second) CAR bindingan antigen of a WBC, and the first CAR binds an antigen of a solidtumor. In embodiments, the solid tumor antigen is tMUC 1, PRLR, CLCA1,MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207,SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3,KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16,MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1,VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, PSCA, or EGFR, and the Bcell antigen is CD19, CD20, CD22, or BCMA.

In embodiments, the CAR comprises an extracellular domain, atransmembrane domain, and an intracellular domain, the extracellulardomain binds an antigen.

In embodiments, the intracellular domain comprises a co-stimulatorydomain that comprises an intracellular domain of a co-stimulatorymolecule selected from the group consisting of CD27, CD28, 4-1BB, OX40,CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and any combination thereof.

In embodiments, the antigen is Epidermal growth factor receptor (EGFR),Variant III of the epidermal growth factor receptor (EGFRvIII), Humanepidermal growth factor receptor 2 (HER2), Mesothelin (MSLN),Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen(CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.

In embodiments, the modified cell or T cells comprise a dominantnegative PD-1 mutant such that PD-1/PDI-1 signaling pathway of the cellis interfered.

In embodiments, the therapeutic agent is present in the modified cell ina recombinant DNA construct, in an mRNA, or in a viral vector. Inembodiments, the modified cell comprises a therapeutic agent mRNAencoding the therapeutic agent, and the mRNA is not integrated into thegenome of the modified cell. In embodiments, the therapeutic agent mRNAmay be introduced (e.g., electroporated) into the modified cell suchthat the expression and/or secretion of the therapeutic agent istransient. Synthetic mRNAs can be injected to achieve transient geneexpression. For example, the therapeutic agent supplied by the mRNA isshort-lived such that the release of the therapeutic agent iscontrollable, especially for proinflammatory cytokines such as: IFN-γ,IL-4, IL-10, TNFα, IL-8, IL-5, IL-6, GM-CSF, and MIP-Iα.

In embodiments, the therapeutic agent comprises or is at least onelisted in Table 2. In embodiments, the therapeutic agent comprises orhas the sequence listed in Table 2.

In embodiments, the modified cell includes a nucleic acid sequencecomprising the isolated nucleic acids described herein, wherein theisolated nucleic acid includes a promoter comprising a binding site fora transcription modulator (e.g., transcription factors) that modulatesthe expression of the therapeutic agent in the cell. Examples of theisolated nucleic acid sequence are provided in Table 2-4. Theseconstructs may be placed into vectors (e.g., lentiviral vectors) eitherin a forward or reverse direction. In embodiments, the transcriptionmodulator includes Hif1a, NFAT, FOXP3, and/or NFkB. In embodiments, thepromoter is responsive to the transcription modulator. In embodiments,the promoter is operably linked to the nucleic acid sequence encodingthe therapeutic agent, such that the promoter drives expression of thetherapeutic agent in the cell. In embodiments, the therapeutic agent isligated to a specific promoter such as to induce expression of thetherapeutic agent in a desired condition. The promoter is divided intotwo parts, a specific regulatory region containing a transcriptionfactor binding site, plus a minimal promoter. In embodiments, thepromoter and the binding site includes the sequences listed in Table2-4. More information about NFAT may be found at WO2018006882, which isincorporated herein by reference.

Embodiments relate to an isolated nucleic acid sequence comprising a(first) nucleic acid and an additional (second) nucleic acid sequence,the first nucleic acid encoding a chimeric antigen receptor (CAR), thesecond nucleic acid encoding a therapeutic agent. For example, thetherapeutic agent comprises IL-6 or IFN-γ, or a combination thereof. Forexample, the therapeutic agent comprises IL-15 or IL-12, or acombination thereof. Embodiments relate to a population of CAR cellscomprising the isolated nucleic acid, wherein the CAR cells compriselymphocyte, leukocyte, or PBMC. In embodiments, the population of CARcells comprise the CAR and the therapeutic agent produced in the form ofa polyprotein, which is cleaved to generate separate CAR and therapeuticagent molecules. In embodiments, the polyprotein comprises a cleavablemoiety between the CAR and the therapeutic agent, the cleavable moietycomprises a 2A peptide, the 2A peptide comprising P2A or T2A. Inembodiments, the CAR and the therapeutic agent are each constitutivelyexpressed. In embodiments, the CAR cells comprise: a third nucleic acidsequence encoding a second CAR binding to an antigen that is differentfrom the first CAR. In embodiments, the second CAR binds a solid tumorantigen, and the first CAR binds an antigen of a white blood cell.

Embodiments relate to a pharmaceutical composition comprising thepopulation of the cells including one or more CAR molecules (the CARcells) and one or more therapeutic agents. Embodiments also relate to amethod of inducing or causing T cell response in a subject in needthereof, the method comprising administering an effective amount of thepharmaceutical composition described herein to the subject. Inembodiments, the CAR cell or the modified cell is a T cell, a NK cell, amacrophage or a dendritic cell. In embodiments, the CAR cell or themodified cell is a T cell.

In embodiments, the additional (second) nucleic acid comprises twonucleic acids, one encoding IL6 and one encoding IFN-γ, and the twonucleic acids are connected by an IRES element or another nucleic acidencoding a 2A peptide. In embodiments, the additional (second) nucleicacid comprises the nucleic acid sequence of SEQ ID NOs: 287 or 328, or acombination thereof. In embodiments, expression of the additional(second) nucleic acid is regulated by a conditional expression systemsuch that the therapeutic agent is expressed in response to binding of atarget antigen. In embodiments, expression of the additional nucleicacid sequence is regulated by SynNotch polypeptide.

Embodiments relate to a FC fusion protein associated with a smallprotein (e.g., a cytokine) as described above. In embodiments, thetherapeutic agent may comprise the FC fusion protein. For example,cytokines such as IL15, IFN-γ or IL6 may be linked to one or moreimmunoglobin Fc domains. In embodiments, the Fc domain foldsindependently and may improve the solubility and stability of the smallprotein both in vitro and in vivo. In embodiments, the Fc region allowsfor easy cost-effective purification by protein-G/A affinitychromatography during manufacture. In embodiments, the FC fusion proteinmay be modified to polymerize into well-defined complexes containingmultiple small proteins. In embodiments, the fusion protein may beexpressed and secreted by the modified cell (e.g., a CAR T cell), whichis used to treat a subject with cancer and/or other diseases. Inembodiments, administration of the fusion protein may be combined withtreatment of CAR T cells expressing and secreting the fusion protein.For example, a method for enhancing T cell response and/or treating asubject with cancer or other diseases may comprise administrating afusion protein associated with the small protein (e.g., IFN-γ) to asubject and administrating an effective amount of the composition of apopulation of T cells comprising a CAR and expressing as well assecreting the fusion protein associated with the small protein to thesubject. In embodiments, the administration of the fusion protein mayenhance expansion of the CAR T cells during the early stage of the CAR Ttreatment (e.g., 1, 2, 3, 4, 5, or 6 days after the infusion of the CART cells). For example, the fusion protein may be administrated into thesubject 1, 2, 3, 4, 5, or 6 days after the infusion of the CAR T cells.In embodiments, the method may comprise administrating a fusion proteinassociated with the small protein (e.g., IFN-γ) to a subject andadministrating an effective amount of the composition of a population ofT cells comprising a CAR without expressing or secreting the fusionprotein associated with the small protein to the subject. For example,the fusion protein may be administrated into the subject for apredetermined time. More information about the FC fusion protein may befound at J Immunol 2004; 172:2925-2934 and EMBO Mol Med. 2012 October;4(10): 1015-1028, which are incorporated by reference. More informationabout administration of the therapeutic agent (e.g. cytokines) may befund at J Interferon Cytokine Res. 2019 January; 39(1):6-21, which isincorporated by reference.

Embodiments relate to a modified cell comprising one or more CARs,wherein the cell is engineered to express and secrete one or moretherapeutic agents. For example, the therapeutic agent comprises IL-6 orIFN-γ, or a combination thereof. Embodiments relate to a method ofinducing or enhancing T cell response, treating cancer, or enhancingcancer treatment, the method comprising: administrating an effectiveamount of the pharmaceutical composition of T cells comprising one ormore CARs, wherein the cell is engineered to express and secrete one ormore therapeutic agents. For example, the therapeutic agent comprisesIL-6 or IFN-γ, or a combination thereof. In embodiments, the therapeuticagent is a small protein associated with IL-6 or IFN-γ. For example,administration of IL-15 to a subject may increase concentrations of IL-6and IFN-γ up to 50-fold in the blood of the patient. Embodiments relateto a method of causing or enhancing T cell response, treating cancer, orenhancing cancer treatment, the method comprising: administering aneffective amount of the composition of a population of T cellscomprising a CAR; and administering an effective amount of a therapeuticagent. For example, therapeutic agent comprises IL-6 or IFN-γ, or acombination thereof. In embodiments, the CAR cells, the modified cell,the cell is a T cell, a NK cell, a macrophage or a dendritic cell. Forexample, the CAR cells, the modified cell, the cell is a T cell.Embodiments relate to a method for enhancing T cell response and/ortreating a subject with cancer or other diseases may compriseadministrating a therapeutic agent (e.g., a recombinant or native IFN-γ)to a subject and administrating an effective amount of a compositioncomprising a population of T cells comprising a CAR and expressing aswell as secreting one or more therapeutic agents in the subject. Inembodiments, the therapeutic agent may enhance expansion of the CAR Tcells during the early stage of the CAR T treatment (e.g., 1, 2, 3, 4,5, or 6 days after the infusion of the CAR T cells). For example, thetherapeutic agent may be administrated into the subject 1, 2, 3, 4, 5,or 6 days after the infusion of the CAR T cells. In embodiments, themethod may comprise administrating a therapeutic agent to a subject andadministrating an effective amount of the composition of a population ofT cells comprising a CAR without expressing or secreting the therapeuticto the subject. For example, the therapeutic agent may be administratedinto the subject for a predetermined time. In embodiments, thetherapeutic agent may be modified such that the biological and/orpharmacological properties of the therapeutic agent may be enhanced. Forexample, the hybrid FC fusion technology may be implemented to thesolubility and/or stability of an active ingredient of the therapeuticagent.

In embodiments, the therapeutic agent may be isolated, synthetic,native, or recombinant human cytokines. For example, Recombinant humanIL-15 may be administered as a daily bolus infusion for predeterminedtime days at 3 mcg/kg/day and 1 mcg/kg/day. Recombinant human IFN-γ maybe administered at a dose of 2 million units daily for 5 days per weekover predetermined time. In embodiments, the administering the effectiveamount of the therapeutic agent comprises administering an effectiveamount of the therapeutic agent such that concentrations of IL-6 and/orIFN-γ in the blood of the subject may increase 5-1000 times (e.g., 50times). For example, the therapeutic agent comprises IL-15.

In embodiments, T cell response is enhanced as compared to theadministration of T cells that do not express or secrete the therapeuticagent, or the T cell response is enhanced as compared to theadministration of CAR T cells without the administration of therapeuticagent.

In embodiments, expression and/or secretion of the therapeutic agent isregulated by an inducible expression system and/or the modified cellcomprises a nucleic acid sequence encoding an inducible suicide system.In embodiments, the inducible expression system is the rtTA-TRE system.In embodiments, the inducible suicide system is an HSV-TK system or aninducible caspase-9 system.

In embodiments, the modified T cells express and/or secrete the one ormore therapeutic agents in response to the activation of the modified Tcells. Such conditional expression and/or secretion may be implementedin various manners. The expression and/or secretion of the one or moretherapeutic agents in the modified cell may be modulated by atranscription modulator (e.g., NFAT). TRUCKs (T cells redirected foruniversal cytokine killing), CAR-redirected T cells, may be used toexpress and/or secrete the one or more therapeutic agents when these Tcells are activated. The expression and/or secretion of the one or moretherapeutic agents in the modified cell may also be regulated by aSynNotch polypeptide.

In embodiments, a range of concentration values of IL6 is 60 to 5000pg/ml, 200-5000 pg/ml, or 2000-5000 pg/ml in the blood of the subject.In embodiments, a range of concentration values IFN-γ is 20 to 5000pg/ml, 200 to 5000 pg/ml, or 500 to 5000 pg/ml in the blood of thesubject. In embodiments, the administering an effective amount of thetherapeutic agent comprises intravenous delivery of an amount of humanIL-6 in the range of about 0.5-50 ug per kilogram of body weight. Inembodiments, the modified cell expresses the therapeutic agent such thatconcentrations of IL-6 and/or IFN-γ in the blood of the subject mayincrease 5-1000 times (e.g., 50 times). For example, the therapeuticagent comprises IL-15. More detailed information about IFN-γ clinicaluses may be found at Cancer Med. 2018, 7: 4509-4516, which isincorporated by reference.

In embodiments, the modified cells or the T cells comprise an additional(second) CAR binding an antigen of a WBC, and the CAR binds an antigenof a solid tumor. In embodiments, the solid tumor antigen is tMUC 1,PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21,TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR,SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3,ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Ra2,Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII,PSCA, or EGFR, and the B cell antigen is CD19, CD20, CD22, or BCMA.

In embodiments, the modified cells or the T cells comprise a dominantnegative PD-1. In embodiments, the modified cell or the T cells comprisea modified PD-1 lacking a functional PD-1 intracellular domain.

In embodiments, the CAR comprises an extracellular domain, atransmembrane domain, and an intracellular domain, the extracellulardomain binds an antigen. In embodiments, the intracellular domaincomprises a co-stimulatory domain that comprises an intracellular domainof a co-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and one combination thereof. In embodiments, the antigen is Epidermalgrowth factor receptor (EGFR), Variant III of the epidermal growthfactor receptor (EGFRvIII), Human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA),Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX(CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125),Cluster of differentiation 133 (CD133), Fibroblast activation protein(FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folatereceptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3,CD5, B-Cell Maturation Antigen (BCMA), or CD4.

In embodiments, the therapeutic agent is present in the modified cell ina recombinant DNA construct, in an mRNA, or in a viral vector. Inembodiments, the modified cell comprises a therapeutic agent mRNAencoding the therapeutic agent, and the mRNA is not integrated into thegenome of the modified cell. In embodiments, the modified cell comprisesa nucleic acid sequence comprising a promoter which comprises a bindingsite for a transcription modulator that modulates the expression and/orsecretion of the therapeutic agent in the cell. In embodiments, thetranscription modulator includes Hif1a, NFAT, FOXP3, and/or NFkB. Inembodiments, the promoter is responsive to the transcription modulator.In embodiments, the promoter is operably linked to the nucleic acidsequence encoding the therapeutic agent such that the promoter drivesexpression and/or secretion of the therapeutic agent in the cell. Inembodiments, the promoter comprises at least one of SEQ ID Nos: 323-325.

In embodiments, the CAR cells, the modified cell, the cell is a T cell,a NK cell, a macrophage or a dendritic cell. For example, the CAR cells,the modified cell, the cell is a T cell.

In embodiments, the population of cells described herein is used inautologous CAR T cell therapy. In embodiments, the CAR T cell therapy isallogenic CAR T cell therapy, TCR T cell therapy, and NK cell therapy.

CAR Molecules

In addition to the embodiments described above, the present disclosuredescribes isolated nucleic acids encoding at least two different antigenbinding domains. In embodiments, there is a first antigen binding domainthat binds an antigen on the surface of a WBC, and there is a secondantigen binding domain that binds an antigen on a tumor that isdifferent from the antigen on the surface of a WBC. The first antigenbinding domain functions to expand the cells that it is introduced into,while the second antigen binding domain functions to inhibit the growthof or kill tumor cells containing the target tumor antigen upon bindingto the target antigen. In embodiments, an isolated nucleic aciddescribed herein encodes both the first and second antigen bindingdomains on the same nucleic acid molecule. In embodiments, the twoantigen binding domains are encoded by two separate nucleic acidmolecules. For example, a first nucleic acid encodes a first antigenbinding domain and a second nucleic acid encodes a second antigenbinding domain.

In embodiments, the present disclosure describes nucleic acids encodinga first antigen binding domain of a binding molecule and a secondantigen binding domain of a binding molecule, wherein first antigenbinding domain binds a cell surface molecule of a WBC, and the secondantigen binding domain binds an antigen different from the cell surfacemolecule of the WBC. In embodiments, the second binding domain does notbind a B cell marker. In embodiments, the second binding domain includesa scFv comprising an amino acid sequence of SEQ ID No: 264 or 265. Forexample, the second antigen binding domain is on a CAR having one of theamino acid sequences of SEQ ID Nos: 271-277.

In embodiments, the first and second antigen binding domains can be ontwo different binding molecules (first and second binding molecules)such as a first CAR and a second CAR. As an example, a first CARincludes an extracellular binding domain that binds a marker on thesurface of a B cell, and a second CAR includes an extracellular bindingdomain that binds a target antigen of a tumor cell. In embodiments, thefirst CAR and second CAR are encoded by different nucleic acids. Inembodiments, the first CAR and second CAR are two different bindingmolecules but are encoded by a single nucleic acid.

In embodiments, the two different antigen binding domains can be on thesame binding molecule, for example on a bispecific CAR, and encoded by asingle nucleic acid. In embodiments, the bispecific CAR can have twodifferent scFv molecules joined together by linkers.

In embodiments, the two different antigen binding domains can be on aCAR and a T cell receptor (TCR) and are encoded by separate nucleicacids. The binding domain of a TCR can target a specific tumor antigenor tumor marker on the cell of a tumor. In embodiments the TCR bindingdomain is a TCR alpha binding domain or TCR beta binding domain thattargets a specific tumor antigen. In embodiments, the TCR comprises theTCRγ and TCRδ chains or the TCRα and TCRβ chains. The present disclosurealso describes vectors including the isolated nucleic acids describedabove. In embodiments, a single vector contains the isolated nucleicacid encoding the first CAR and second CAR or TCR. In embodiments, afirst vector contains the first nucleic acid encoding a first CAR, asecond vector contains the nucleic acid encoding the second CAR or TCR.In embodiments, the vector comprises a bispecific CAR including at leasttwo antigen binding domains.

Moreover, the present disclosure describes cells comprising the isolatednucleic acids or vectors described above. The cells have been introducedwith the isolated nucleic acids or vectors described herein and expressat least two more binding domains. In embodiments, the cells include twoor more different binding domains, a first antigen binding domain and asecond antigen binding domain, wherein the first antigen binding domainbinds a cell surface molecule of a WBC, and the second antigen bindingdomain binds an antigen different from the cell surface molecule of aWBC. Further, the present disclosure describes compositions including apopulation of the cells described herein. In embodiments, the cells areperipheral blood mononuclear cells (PBMCs) such as lymphocytes. Inembodiments, the lymphocytes are T cells, NK cell, or dendritic cells.

The present disclosure also describes methods of culturing cellsdescribed above. The methods described herein includes obtaining a cellcomprising a first antigen binding domain and a second antigen bindingdomain, wherein the first antigen binding domain binds a cell surfacemolecule of a WBC, and the second antigen binding domain binds anantigen different from the cell surface molecule of the WBC; andculturing the cell in the presence of an agent derived from a cellsurface molecule of the WBC or from an antigen to which the secondantigen binding domain binds. In embodiments, the agent is anextracellular domain of a cell surface molecule of a WBC.

The present disclose describes methods for in vitro cell preparation,wherein the method includes providing cells; introducing one or morenucleic acids encoding a first antigen binding domain and a secondantigen binding domain into the cells, wherein the first antigen bindingdomain binds a cell surface molecule of a WBC, and the second antigenbinding domain binds an antigen different from the cell surface moleculeof the WBC; and culturing the cells in the presence of an agent derivedfrom the cell surface molecule of the WBC or from an antigen to whichthe second antigen binding domain binds.

The present disclosure describes using the prepared cells preparation toenhance T cell expansion in a subject having cancer. In embodiments, themethod comprises introducing a plurality of nucleic acids into T cells,the plurality of nucleic acids encoding a chimeric antigen receptor(CAR) binding a solid tumor antigen and encoding a CAR binding a B cellantigen, at least a portion of the T cells comprising the CAR bindingthe solid tumor antigen and the CAR binding the B cell antigen; andadministering an effective amount of the T cells to the subject. The Tcell expansion is enhanced or the number of T cells is increased in thesubject as compared to a subject that is administered with T cellscomprising the plurality of nucleic acids encoding only one CAR.

Additionally, the present disclosure describes methods for introducingand/or enhancing lymphocyte (T cell) response in a subject. Embodimentsdescribed herein involve a mechanism that expands lymphocytes and amechanism that relates to binding of an antigen on a CAR to a tumorcell. In embodiments, the first mechanism involves a molecule associatedwith a signal that is involved in expanding the lymphocytes in asubject, and an additional mechanism involves a molecule associated witha signal directed to binding, inhibiting the growth of, or killing atumor cell in the subject. For example, the first mechanism includes aCAR binding to an antigen associated with blood, such as blood cells andblood plasma, or non-essential tissues, and the additional mechanismincludes a CAR or TCR targeting an antigen associated with the tumorcell. Examples of non-essential tissues include the mammary gland,colon, gastric gland, ovary, blood components, such WBC, and thyroid. Inembodiments, the first mechanism involves a first binding domain of amolecule, and the additional mechanism involves a second domain of amolecule. In embodiments, the first mechanism and the additionalmechanism are performed by the same molecule or by separate molecules.In particular embodiments, the mechanism involves a cell expressing anantigen associated with a tumor cell, and the additional mechanisminvolves a lymphocyte having an antigen binding domain.

The methods described herein involves lymphocytes including an expansionmolecule and a function molecule. In embodiments, the expansion moleculeexpands the lymphocytes in a subject, and/or the function moleculeinhibits the growth of or kills a tumor cell in the subject. Inembodiments, the expansion molecule and the function molecule are on asingle CAR molecule, for example a bispecific CAR molecule. Inembodiments, the expansion molecule and the function molecule are onseparate molecules, for example, CAR and TCR or two different CARs. Theexpansion molecule can include a CAR binding to an antigen associatedwith blood (e.g., blood cells and blood plasma) or non-essentialtissues, and the function molecule can include a CAR or TCR targeting anantigen associated with the tumor cell.

Lymphocyte or T cell response in a subject refers to cell-mediatedimmunity associated with a helper, killer, regulatory, and other typesof T cells. For example, T cell response may include activities such asassisting other WBCs in immunologic processes and identifying anddestroying virus-infected cells and tumor cells. T cell response in thesubject can be measured via various indicators such as a number ofvirus-infected cells and/or tumor cells that T cells kill, the amount ofcytokine that T cells release in co-culturing with virus-infected cellsand/or tumor cells, a level of proliferation of T cells in the subject,a phenotype change of T cells, for example, changes to memory T cells,and a level longevity or lifetime of T cells in the subject.

In embodiments, the method of enhancing T cell response comprisestreating a subject in need thereof, for example, a subject diagnosedwith a tumor. The term tumor refers to a mass, which can be a collectionof fluid, such as blood, or a solid mass. A tumor can be malignant(cancerous) or benign. Examples of blood cancers include chroniclymphocytic leukemia, acute myeloid leukemia, acute lymphoblasticleukemia, and multiple myeloma.

Solid tumors usually do not contain cysts or liquid areas. The majortypes of malignant solid tumors include sarcomas and carcinomas.Sarcomas are tumors that develop in soft tissue cells called mesenchymalcells, which can be found in blood vessels, bone, fat tissues, ligamentlymph vessels, nerves, cartilage, muscle, ligaments, or tendon, whilecarcinomas are tumors that form in epithelial cells, which are found inthe skin and mucous membranes. The most common types of sarcomas includeundifferentiated pleomorphic sarcoma which involves soft tissue and bonecells; leiomyosarcoma which involves smooth muscle cells that line bloodvessels, gastrointestinal tract, and uterus; osteosarcoma which involvesbone cells, and liposarcoma which involves fat cells. Some examples ofsarcomas include Ewing sarcoma, Rhabdomyosarcoma, chondosarcoma,mesothelioma, fibrosarcoma, fibrosarcoma, and glioma.

The five most common carcinomas include adrenocarcinoma which involvesorgans that produce fluids or mucous, such as the breasts and prostate;basal cell carcinoma which involves cells of the outer-most layer of theskin, for example, skin cancer; squamous cell carcinoma which involvesthe basal cells of the skin; and transitional cell carcinoma whichaffects transitional cells in the urinary tract which includes thebladder, kidneys, and ureter. Examples of carcinomas include cancers ofthe thyroid, breast, prostate, lung, intestine, skin, pancreas, liver,kidneys, and bladder, and cholangiocarcinoma.

The methods described herein can be used to treat a subject diagnosedwith cancer. The cancer can be a blood cancer or can be a solid tumor,such as a sarcoma or carcinoma. The method of treating includesadministering an effective amount of T cells comprising a first antigenbinding domain and a second antigen binding domain to the subject toprovide a T-cell response, wherein the first antigen binding domainbinds a cell surface molecule of a WBC, and the second antigen bindingdomain binds an antigen different from the cell surface molecule of theWBC. In embodiments, enhancing the T cell response in the subjectincludes selectively enhancing proliferation of T cell expressing thefirst antigen binding domain and the second antigen binding domain invivo.

In embodiments, the T cells for enhancing T cell response in a subjectincludes administering to the subject, T cells comprising a bispecificCAR including two different binding domains or administering T cellscomprising a first CAR and a second CAR, wherein the first CAR and thesecond CAR, each includes a different antigen binding domain.

In embodiments, methods for enhancing T cell response in a subjectincludes administering a T cell including a CAR molecule and a TCRmolecule. The CAR molecule targets or binds a surface marker of a whiteblood cell, and the TCR molecule binds a marker or an antigen of thetumor that is expressed on the surface or inside the tumor cell.

The present disclosure describes methods of expanding cells expressingan antigen binding domain in vivo. The method includes administering aneffective amount of T cells comprising a first antigen binding domainand a second antigen binding domain to a subject in need thereof,wherein the first antigen binding domain binds a cell surface moleculeof a WBC, and the second antigen binding domain binds an antigendifferent from the cell surface molecule of the WBC. The methods areuseful for expanding or increasing the number of T cells, NK cells,dendritic cells.

In embodiments, the first antigen binding domain is on a first chimericantigen receptor (CAR) and the second antigen binding domain is on asecond CAR or a TCR. For example, the first CAR and the second CAR orTCR include an extracellular antigen binding domain, a transmembranedomain, and a cytoplasmic domain. The cytoplasmic domain of the firstCAR include a co-stimulatory domain and a CD3 zeta domain fortransmitting signals for activation of cellular responses. Inembodiments, the cytoplasmic domain of the first CAR includes one ormore co-stimulatory domains in the absence of a CD3 zeta domain suchthat activation or stimulation of the first CAR expands WBCs, such aslymphocytes, without introducing and/or activating the killing functionof the WBCs. In embodiments, the lymphocytes are T cells.

In embodiments, the first and second antigen binding domains are on thesame CAR (the first CAR), for example, a bispecific CAR with anextracellular antigen binding domain, a transmembrane domain, and acytoplasmic domain. The extracellular antigen binding domain includes atleast two scFvs and at least one of the scFvs function as a firstantigen binding domain for binding a cell surface molecule of a WBC.

In embodiments, the antigen different from the cell surface molecule ofthe WBC is CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30,CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, CD13, B7, CAIX,CD123, CD133, CD171, CD171/L1-CAM, CEA, Claudin 18.2, cMet, CS1, CSPG4,Dectin1, EGFR, EGFR vIII, EphA2, ERBB receptors, ErbB T4, ERBB2, FAP,Folate receptor 1, FITC, Folate receptor 1, FSH, GD2, GPC3, HA-1H/HLA-A2, HER2, IL-11Ra, IL13 receptor a2, IL13R, IL13Rα2 (zetakine),Kappa, Leukemia, LewisY, Mesothelin, MUC1, NKG2D, NY-ESO-1, PSMA, ROR-1,TRAIL-receptor1, or VEGFR2.

In embodiments, the MUC1 is a tumor-exclusive epitope of a human MUC1,and the first CAR and the second CAR or the TCR are expressed asseparate polypeptides. In embodiments, the MUC1 is a tumor form of humanMUC1 (tMUC1).

In embodiments, the first CAR includes a co-stimulatory domain without asignaling domain, such as the CD3 zeta domain, and the MUC1 CAR (secondCAR) comprises the MUC1 binding domain, a transmembrane domain, aco-stimulatory, and a CD3 zeta domain.

As used herein, the term “MUC1” refers to a molecule defined as follows.MUC1 is one of the epithelial mucin family of molecules. MUC1 is atransmembrane mucin glycoprotein that is normally expressed on allglandular epithelial cells of the major organs. In normal cells, MUC1 isonly expressed on the apical surface and is heavily glycosylated withits core proteins sequestered by the carbohydrates. As cells transformto a malignant phenotype, expression of MUC1 increases several folds,and the expression is no longer restricted to the apical surface, but itis found all around the cell surface and in the cytoplasm. In addition,the glycosylation of tumor associated MUC1 is aberrant, with greaterexposure of the peptide core than is found on MUC1 expressed in normaltissues. Little is known regarding the specifics of the aberrantglycosylation.

MUC1 is widely expressed on a large number of epithelial cancers and isaberrantly glycosylated making it structurally and antigenicallydistinct from that expressed by non-malignant cells (see, e.g.,Barratt-Boyes, 1996; Price et al., 1998; Peterson et al., 1991). Thedominant form of MUC1 is a high molecular weight molecule comprising alarge highly immunogenic extracellular mucin-like domain with a largenumber of twenty amino acid tandem repeats, a transmembrane region, anda cytoplasmic tail (Quin et al., 2000; McGucken et al., 1995; Dong etal., 1997).

In most epithelial adenocarcinomas including breast and pancreas, MUC1is overexpressed and aberrantly glycosylated. Adenocarcinoma of thebreast and pancreas not only overexpress MUC1 but also shed MUC1 intothe circulation. High MUC1 serum levels are associated with progressivedisease. MUC1 has been exploited as a prospective biomarker because ofthe complex and heterogeneous nature of the epitopes expressed withinthe antigen. MUC1 synthesized by cancerous tissues (e.g., tumorassociated MUC1) usually displays an aberrant oligosaccharide profile,which gives rise to the expression of neomarkers such as sialyl-Lea(assayed in the CA19-9 test), sialyl-Lex, and sialyl-Tn (TAG-72), aswell as the cryptic epitopes such as Tn.

Several antibodies are being developed against MUC1 for therapeutic use.Pemtumomab (also known as HMFG1) is in Phase III clinical trials as acarrier to deliver the radioisotope Yttrium-90 into tumors in ovariancancer (reviewed in Scott et al., 2012). CA15-3 (also the HMFG1antibody), CA27-29, and CA19-9 are all antibodies to MUC1 that are usedto assess levels of circulating MUC1 in patients with cancer. However,these antibodies have shown limited utility as therapeutic agents or asbiomarkers because they cannot distinguish effectively between MUC1expressed on normal versus transformed tumor epithelia. In other words,none of these antibodies appear to be targeted to a tumor-specific MUC1epitope.

A new antibody that is highly specific for a tumor-specific form of MUC1(tMUC) is designated TAB-004 and is described in U.S. Pat. No. 8,518,405(see also Curry et al., 2013). While Pemtumomab (HMFG1) was developedusing human milk fat globules as the antigen (Parham et al., 1988),TAB-004 was developed using tumors expressing an altered form of MUC1(Tinder et al., 2008). TAB-004 recognizes the altered glycosylatedepitope within the MUC1 tandem repeat sequence. This area is accessiblefor antigenic detection in tMUC but is blocked from antigenic detectionin normal MUC1 by large branches of glycosylation (Gendler, 2001;Mukherjee et al., 2003b; Hollingsworth & Swanson, 2004; Kufe, 2009).Importantly, TAB-004 is different from the epitopes recognized by otherMUC1 antibody and has unique complementary determinant regions (CDRs) ofthe heavy and light chains. The antibody binds the target antigen with ahigh binding affinity at 3 ng/ml (20 pM) and does not bind unrelatedantigens (Curry et al., 2013). Thus, TAB-004 distinguishes betweennormal and tumor form of MUC1 while HMFG1 (Pemtumomab) does not (seeU.S. Pat. No. 8,518,405).

In embodiments, the WBC is a granulocyte, monocyte and or lymphocyte. Inembodiments, the WBC is a B cell.

In embodiments, the first CAR comprises the first antigen bindingdomain, a transmembrane domain, a co-stimulatory domain, and a CD3 zetadomain and/or the second CAR comprises the second antigen bindingdomain, a transmembrane domain, a co-stimulatory domain, and a CD3 zetadomain.

In embodiments, the antigen binding domain is a Fab or a scFv. Inembodiments, the first CAR comprises the amino acid sequence of one ofSEQ ID NO: 5, 6, and 53-58; and the second CAR comprises the amino acidsequence of one of SEQ ID NOs: 5-17, 29, 33, 37, 71, and 72, or theamino acid sequence encoded by the nucleic acid sequence of one of SEQID Nos: 41, 45, 63, 67, and 68. In embodiments, a nucleic acid sequenceencoding the first CAR comprises the nucleic acid sequence of SEQ ID NO:59 or 60, and a nucleic acid sequence encoding the second CAR comprisesthe nucleic acid sequence of SEQ ID NO: 61. In embodiments, the isolatednucleic acid comprises one of the nucleic acid sequence of SEQ ID NO:62-69. In embodiments, the first CAR and the second CAR are expressed asseparate polypeptides.

In embodiments, the first antigen binding domain is on a CAR and thesecond antigen binding domain is on a T Cell Receptor (TCR). Inembodiments, the TCR is a modified TCR. In embodiments, the TCR isderived from spontaneously occurring tumor-specific T cells in patients.In embodiments, the TCR binds a tumor antigen. In embodiments, the tumorantigen comprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1.

In embodiments, a T cell clone that expresses a TCR with high affinityfor the target antigen may be isolated. Tumor-infiltrating lymphocytes(TILs) or peripheral blood mononuclear cells (PBMCs) can be cultured inthe presence of antigen-presenting cells (APCs) pulsed with a peptiderepresenting an epitope known to elicit a dominant T cell response whenpresented in the context of a defined HLA allele. High-affinity clonesmay be then selected on the basis of MHC-peptide tetramer stainingand/or the ability to recognize and lyse target cells pulsed with lowtitrated concentrations of cognate peptide antigen. After the clone hasbeen selected, the TCRα and TCR chains or TCRγ and TCRδ chains areidentified and isolated by molecular cloning. For example, for TCRα andTCRβ chains, the TCRα and TCRβ gene sequences are then used to generatean expression construct that ideally promotes stable, high-levelexpression of both TCR chains in human T cells. The transductionvehicle, for example, a gammaretrovirus or lentivirus, can then begenerated and tested for functionality (antigen specificity andfunctional avidity) and used to produce a clinical lot of the vector. Analiquot of the final product can then be used to transduce the target Tcell population (generally purified from patient PBMCs), which isexpanded before infusion into the patient.

Various methods may be implemented to obtain genes encodingtumor-reactive TCR. More information is provided in Kershaw et al., ClinTransl Immunology. 2014 May; 3(5): e16. In embodiments, specific TCR canbe derived from spontaneously occurring tumor-specific T cells inpatients. Antigens included in this category include the melanocytedifferentiation antigens MART-1 and gp100, as well as the MAGE antigensand NY-ESO-1, with expression in a broader range of cancers. TCRsspecific for viral-associated malignancies can also be isolated, as longas viral proteins are expressed by transformed cells. Malignancies inthis category include liver and cervical cancer, associated withhepatitis and papilloma viruses, and Epstein-Barr virus-associatedmalignancies. In embodiments, target antigens of the TCR may include CEA(e.g., for colorectal cancer), gp100, MART-1, p53 (e.g., for Melanoma),MAGE-A3 (e.g., Melanoma, esophageal and synovial sarcoma), NY-ESO-1(e.g., for Melanoma and sarcoma as well as Multiple myelomas).

In embodiments, a binding domain of the first CAR binds CD19, and abinding domain of the second CAR binds tumor associated MUC1. Inembodiments, the binding domain of the second CAR comprises: (i) a heavychain complementary determining region 1 comprising the amino acidsequence of SEQ ID: 76 or 85, a heavy chain complementary determiningregion 2 comprising the amino acid sequence of SEQ ID: 77 or 86, and aheavy chain complementary determining region 3 comprising the amino acidsequence of SEQ ID: 78 or 87; and (ii) a light chain complementarydetermining region 1 comprising the amino acid sequence of SEQ ID: 73 or82, a light chain complementary determining region 2 comprising theamino acid sequence of TRP-ALA-SER (WAS) or SEQ ID: 83, and a lightchain complementary determining region 3 comprising the amino acidsequence of SEQ ID: 75 or 84.

In embodiments, the binding domain of the second CAR comprises: (i) aheavy chain complementary determining region 1 comprising the amino acidsequence of SEQ ID: 76, a heavy chain complementary determining region 2comprising the amino acid sequence of SEQ ID: 77, and a heavy chaincomplementary determining region 3 comprising the amino acid sequence ofSEQ ID: 78; and (ii) a light chain complementary determining region 1comprising the amino acid sequence of SEQ ID: 73, a light chaincomplementary determining region 2 comprising the amino acid sequence ofTRP-ALA-SER (WAS), and a light chain complementary determining region 3comprising the amino acid sequence of SEQ ID: 75.

In embodiments, the binding domain of the second CAR comprises: (i) aheavy chain complementary determining region 1 comprising the amino acidsequence of SEQ ID: 85, a heavy chain complementary determining region 2comprising the amino acid sequence of SEQ ID: 86, and a heavy chaincomplementary determining region 3 comprising the amino acid sequence ofSEQ ID: 87; and (ii) a light chain complementary determining region 1comprising the amino acid sequence of SEQ ID: 82, a light chaincomplementary determining region 2 comprising the amino acid sequence ofSEQ ID: 83, and a light chain complementary determining region 3comprising the amino acid sequence of SEQ ID: 84. In embodiments, thebinding domain of the first CAR comprises the amino acid sequence of SEQID: 5 or 6. In embodiments, the binding domain of the second CARcomprises one of the amino acid sequences of SEQ ID: 70-72 and 79-81.

In embodiments, the first CAR comprises the first antigen bindingdomain, a transmembrane domain, a co-stimulatory domain, and a CD3 zetadomain and/or the second CAR comprises the second antigen bindingdomain, a transmembrane domain, a co-stimulatory domain, and a CD3 zetadomain.

In embodiments, the first CAR and the second CAR are expressed asseparate polypeptides.

In embodiments, the cytoplasmic domain or the transmembrane domain ofthe second CAR is modified such that the second CAR is capable ofactivating the modified T cell via cells expressing CD19 withoutdamaging the cells expressing CD19.

Embodiments described herein relate to a bispecific chimeric antigenreceptor, comprising: a first antigen binding domain, a second antigenbinding domain, a cytoplasmic domain, and transmembrane domain, whereinthe first antigen binding domain recognizes a first antigen, and thesecond antigen binding domain recognizes a second antigen, the firstantigen is different from the second antigen.

In embodiments, the first antigen and the second antigen do not expresson the same cell. In embodiments, the first antigen is an antigen of ablood component, and the second antigen is an antigen of a solid tumor.

Blood cells refer to red blood cells (RBCs), white blood cells (WBCs),platelets, or other blood cells. For example, RBCs are blood cells ofdelivering oxygen (O₂) to the body tissues via the blood flow throughthe circulatory system. Platelets are cells that are involved inhemostasis, leading to the formation of blood clots. WBCs are cells ofthe immune system involved in defending the body against both infectiousdisease and foreign materials. There are a number of different types andsub-types of WBCs and each has a different role to play. For example,granulocytes, monocytes, and lymphocytes are 3 major types of whiteblood cell. There are three different forms of granulocytes:Neutrophils, Eosinophils, Basophils.

A cell surface molecule of a WBC refers to a molecule expressed on thesurface of the WBC. For example, the cell surface molecule of alymphocyte may include CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16,CD56, and CD30. The cell surface molecule of a B cell may include CD19,CD20, CD22, BCMA. The cell surface molecule of a monocyte may includeCD14, CD68, CD11b, CD18, CD169, and CD1c. The cell surface molecule ofgranulocyte may include CD33, CD38, CD138, and CD13.

In embodiments, the first antigen is CD19, and the second antigen is atumor associated MUC1. In embodiments, the first antigen binding domaincomprises one of the amino acid sequences of SEQ ID: 5 and 6. Inembodiments, the second antigen binding domain comprises one of theamino acid sequence of SEQ ID: 70-72 and 79-81.

In embodiments, the present disclosure describes a method of enhancing Tcell response in a subject or treating a tumor of the subject, themethod comprising: administering an effective amount of modified T cellto the subject to provide a T cell response such that the CART cell isexpanded in the blood of the subject via cells expressing CD19.

In embodiments, the tumor associated MUC1 is expressed on tumor cells,but not on corresponding non-malignant cells. In embodiments, a scFvagainst the tumor associated MUC1 directly interacts with ano-glycosylated GSTA motif (SEQ ID NO. 88).

Embodiments described herein relate to a cell comprising the bispecificCAR and to an isolated nucleic acid encoding the bispecific CAR.

In embodiments, the present disclosure describes a method of in vivocell expansion. In embodiments, the method may include administering aneffective amount of T cell comprising a CAR to the subject to provide aT cell response; and administering an effective amount of presentingcells (e.g., T cells) expressing a soluble agent that an extracellulardomain of the CAR recognizes. In embodiments, the method may beimplemented to enhance T cell response in a subject. The method mayinclude administering an effective amount of T cell comprising a CAR tothe subject to provide a T cell response and administering an effectiveamount of presenting cells expressing a soluble agent that anextracellular domain of the CAR recognizes to enhance the T cellresponse in the subject. In certain embodiments, the presenting cellsare T cells, dendritic cells, and/or antigen presenting cells. Incertain embodiments, the enhancing T cell response in the subject mayinclude selectively enhancing proliferation of T cell comprising theCAR. In embodiments, the method may be used to enhance treatment of acondition on a subject using CAR T cells. The method may includeadministering a population of cells that express an agent oradministering an agent that is formulated as a vaccine. In theseinstances, the CART cells include a nucleic acid that encodes a CAR, andan extracellular domain of the CAR recognize the agent. In embodiments,the method may be implemented to enhance proliferation of CAR T cells ina subject having a disease. The method may include preparing CART cellscomprising a CAR; administering an effective amount of the CART cells tothe subject; introducing, into cells, a nucleic acid encoding an agentthat an extracellular domain of the CAR recognizes; and administering aneffective amount of the cells (introduced with the nucleic acid encodingthe agent) to the subject. In embodiments, the T cell expansion orincreased in the number of T cells may be measured based on an increasein copy number of CAR molecules in genomic DNA of the T cells. Inembodiments, the T cell expansion or increased in the number of T cellsmay be measured based on flow cytometry analysis on molecules expressedon the T cells.

Embodiments described herein relate to an isolated T cell comprising afirst CAR, and a second CAR, wherein an antigen binding domain of thefirst CAR binds an antigen such as CD19, CD33, CD14, and BCMA, and anantigen binding domain of the second CAR binds a tumor associated MUC.In embodiments, the tumor associated MUC is MUC1 or MUC2. Embodimentsdescribed herein relate to a composition comprising a population of theisolated T cells and to a method of enhancing T cell response in asubject or treating a tumor of the subject, the method comprising:administering an effective amount of the isolated T cell.

In embodiments, the first CAR comprises the amino acid sequence of SEQID NO: 207, and the second CAR comprises the amino acid sequence of SEQID: 202. In embodiments, the first CAR comprises the amino acid sequenceof SEQ ID NO: 203, 207, 216, or 219, and the second CAR comprises theamino acid sequence of SEQ ID: 202 or 205. In embodiments, the antigenbinding domain of the second CAR comprises the amino acid sequence ofSEQ ID NO: 70. In embodiments, the antigen binding domain of the secondCAR comprises the amino acid sequence of SEQ ID NO: 5 or 6. Inembodiments, the isolated T cell comprises a nucleic acid sequence ofSEQ ID NO: 201, 204, 206, 208, 215, 217, 218, or 220. In embodiments,each of the first CAR and the second CAR comprises an antigen bindingdomain, a transmembrane domain, and a cytoplasmic domain.

In embodiments, the cytoplasmic domain comprises a co-stimulatory domainand a CD3 zeta domain.

In embodiments, the isolated T cell comprises a dominant negativevariant of a receptor of programmed death 1 (PD-1), cytotoxic Tlymphocyte antigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), Tcell immunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3(LAG-3), T cell immunoreceptor with Ig and ITIM domains (TIGIT),leukocyte-associated immunoglobulin-like receptor 1 (LAIRI), naturalkiller cell receptor 2B4 (2B4), or CD 160. In embodiments, the isolatedT cell comprises a reduced amount of TCR, as compared to thecorresponding wide-type T cell. Dominant negative mutations have analtered gene product that acts antagonistically to the wild-type allele.These mutations usually result in an altered molecular function (ofteninactive) and are characterized by a dominant or semi-dominantphenotype.

The present disclosure describes pharmaceutical compositions. Thepharmaceutical compositions include one or more of the following: CARmolecules, TCR molecules, modified CAR T cells, modified cellscomprising CAR or TCR, modified cells, nucleic acids, and vectorsdescribed above. Pharmaceutical compositions are administered in amanner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-tumor effectiveamount”, “a tumor-inhibiting effective amount”, or “therapeutic amount”is indicated, the precise amount of the compositions of the presentdisclosure to be administered can be determined by a physician withconsideration of individual differences in age, weight, tumor size,extent of infection or metastasis, and condition of the patient(subject). It can be stated that a pharmaceutical composition comprisingthe T cells described herein may be administered at a dosage of 10⁴ to10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. T cell compositionsmay also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly. In certain embodiments, it may be desired toadminister activated T cells to a subject and then subsequently redrawthe blood (or have apheresis performed), collect the activated andexpanded T cells, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 cc to 400 cc. In certain embodiments, T cells areactivated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc,80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocols, may select out certainpopulations of T cells.

The administration of the pharmaceutical compositions described hereinmay be carried out in any convenient manner, including by aerosolinhalation, injection, ingestion, transfusion, implantation, ortransplantation. The compositions described herein may be administeredto a patient subcutaneously, intradermally, intratumorally,intranodally, intramedullary, intramuscularly, by intravenous (i. v.)injection, or intraperitoneally. In embodiments, the T cell compositionsdescribed herein are administered to subjects by intradermal orsubcutaneous injection. In embodiments, the T cell compositions of thepresent disclosure are administered by i.v. injection. The compositionsof T cells may be injected directly into a tumor, lymph node, or site ofinfection. In embodiments, cells activated and expanded using themethods described herein, or other methods known in the art where Tcells are expanded to therapeutic levels, are administered to patientsin conjunction with (e.g., before, simultaneously or following) anynumber of relevant treatment modalities, including but not limited totreatment with agents for antiviral therapy, cidofovir andinterleukin-2, Cytarabine (also known as ARA-C); or natalizumabtreatment for MS patients; or efalizumab treatment for psoriasispatients or other treatments for PML patients. In further embodiments,the T cells described herein can be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAM PATH, anti-CD3 antibodies orother antibody therapies, cytoxin, fludaribine, cyclosporin, FK506,rapamycin, mycophenolic acid, steroids, FR901228, cytokines, andirradiation. These drugs inhibit either the calcium dependentphosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun73:316-321, 1991; Bierer et al., Curr. Opin. Immun 5:763-773, 1993;Isoniemi (supra)). In embodiments, the cell compositions describedherein are administered to a subject in conjunction with (e.g., before,simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In embodiments, the cell compositions describedherein are administered following B-cell ablative therapy. For example,agents that react with CD20, e.g., Rituxan may be administered topatients. In embodiments, subjects may undergo standard treatment withhigh dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentdisclosure. In embodiments, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a subject inneed thereof will vary with the precise nature of the condition beingtreated and the recipient of the treatment. The scaling of dosages forhuman administration can be performed according to art-acceptedpractices by a physician depending on various factors.

Additional information on the methods of cancer treatment usingengineered or modified T cells is provided in U.S. Pat. No. 8,906,682,incorporated by reference in its entirety.

Embodiments described herein relate to an in vitro method for preparingmodified cells. The method may include obtaining a sample of cells froma subject. For example, the sample may include T cells or T cellprogenitors. The method may further include transfecting the sample ofcells with a DNA encoding at least a CAR and culturing the population ofCAR cells ex vivo in a medium that selectively enhances proliferation ofCAR-expressing T cells.

In embodiments, the sample is a cryopreserved sample. In embodiments,the sample of cells is from umbilical cord blood or a peripheral bloodsample from the subject. In embodiments, the sample of cells is obtainedby apheresis or venipuncture. In embodiments, the sample of cells is asubpopulation of T cells.

Tables 2-4

TABLE 2 SEQ SEQ SEQ ID ID ID Name NO: Name NO: Name No: SP 1 UPK2 101Construct of MUC1- 201 5E5-A-IRES-CD19-A Hinge & 2 ADAM12 102 CAR 1 ofMUC1-5E5- 202 transmembrane A-IRES-CD19-A domain Co-stimulatory 3SLC45A3 103 CAR 2 of MUC1-5E5- 203 domain A-IRES-CD19-A CD3-zeta 4 ACPP104 Construct of MUC1- 204 5E5-B-IRES-CD19-A scFV Humanized 5 MUC21 105CAR 1 of MUC1-5E5- 205 CD19 B-IRES-CD19-A ScFV CD19 6 MUC16 106 CAR 2 ofMUC1-5E5- 203 B-IRES-CD19-A scFv FZD10 7 MS4A12 107 Construct of MUC1-206 5E5-A-IRES-CD19-B scFv TSHR 8 ALPP 108 CAR 1 of MUC1-5E5- 202A-IRES-CD19-B scFv PRLR 9 SLC2A14 109 CAR 2 of MUC1-5E5- 207A-IRES-CD19-B scFv Muc 17 10 GS1-259H13.2 110 Construct of MUC1- 2085E5-B-IRES-CD19-B scFv GUCY2C 11 ERVFRD-1 111 CAR 1 of MUC1-5E5- 205B-IRES-CD19-B scFv CD207 12 ADGRG2 112 CAR 2 of MUC1-5E5- 207B-IRES-CD19-B Prolactin (ligand) 13 ECEL1 113 Construct of MUC1-2- 209A-IRES-CD19-A scFv CD3 14 CHRNA2 114 CAR 1 of MUC1-2-A- 210 IRES-CD19-AscFv CD4 15 GP2 115 CAR 2 of MUC1-2-A- 203 IRES-CD19-A scFv CD4-2 16PSG9 116 Construct of MUC1-2- 211 B-IRES-CD19-A scFv CD5 17 SIGLEC15 117CAR 1 of MUC1-2-B- 212 IRES-CD19-A CD19 antigen 18 SLC6A3 118 CAR 2 ofMUC1-2-B- 203 IRES-CD19-A FZD10 antigen 19 KISS1R 119 Construct ofMUC1-2- 213 A-IRES-CD19-B TSHR antigen 20 QRFPR 120 CAR 1 of MUC1-2-A-210 IRES-CD19-B PRLR antigen 21 GPR119 121 CAR 2 of MUC1-2-A- 207IRES-CD19-B Muc 17 antigen 22 CLDN6 122 Construct of MUC1-2- 214B-IRES-CD19-B GUCY2C antigen 23 SP-2 123 CAR 1 of MUC1-2-B- 212IRES-CD19-B CD207 antigen 24 Linker-2 124 CAR 2 of MUC1-2-B- 207IRES-CD19-B CD3 antigen 25 Hinge-2 125 Construct of MUC1- 2155E5-A-IRES-hCD19-A CD4 antigen 26 TM-2 126 CAR 1 of MUC1-5E5- 202A-IRES-hCD19-A CD5 antigen 27 4-1BB-2 127 CAR 2 of MUC1-5E5- 216A-IRES-hCD19-A CAR CD19 nucleic 28 CD3 zeta-2 128 Construct of MUC1- 217acid 5E5-B-IRES-hCD19-A Hinge & TM 29 CLDN6-CAR-1 129 CAR 1 of MUC1-5E5-205 domain B B-IRES-hCD19-A Hinge & TM 30 ScFv CLDN6-CAR-1 130 CAR 2 ofMUC1-5E5- 216 domain A B-IRES-hCD19-A Hinge & TM 31 ScFv VL CLDN6- 131Construct of MUC1- 218 domain D CAR-1 5E5-A-IRES-hCD19-B Hinge & TM 32ScFv VH CLDN6- 132 CAR 1 of MUC1-5E5- 202 domain C CAR-1 A-IRES-hCD19-BHinge domain D 33 CLDN6-CAR-2 133 CAR 2 of MUC1-5E5- 219 A-IRES-hCD19-BHinge domain C 34 ScFv CLDN6-CAR-2 134 Construct of MUC1- 2205E5-B-IRES-hCD19-B Hinge domain B 35 ScFv VL CLDN6- 135 CAR 1 ofMUC1-5E5- 205 CAR-2 B-IRES-hCD19-B Hinge domain A 36 ScFv VH CLDN6- 136CAR 2 of MUC1-5E5- 219 CAR-2 B-IRES-hCD19-B TM domain D 37 CLDN6-CAR-3137 Construct of MUC1-2- 221 A-IRES-hCD19-A TM domain A 38 scFvCLDN6-CAR-3 138 CAR 1 of MUC1-2-A- 210 IRES-hCD19-A CD19 extracellular39 scFv VL CLDN6-CAR- 139 CAR 2 of MUC1-2-A- 216 domain 3 IRES-hCD19-ATM domain C or B 40 scFv VH CLDN6- 140 Construct of MUC1-2- 222 CAR-3B-IRES-hCD19-A WTCD3zeta 41 CLDN6-CAR-4 141 CAR 2CAR 1 of 212MUC1-2-B-IRES- hCD19-A WTCD3zeta- 42 scFv CLDN6-CAR-4 142 Construct ofMUC1-2- 216 BCMACAR full B-IRES-hCD19-A length BCMA 43 scFv VLCLDN6-CAR- 143 Construct of MUC1-2- 223 4 A-IRES-hCD19-B BCMA CAR vector44 scFv VH CLDN6- 144 CAR 1 of MUC1-2-A- 210 CAR-4 IRES-hCD19-B BCMA CARvector 45 SIGLEC-15-CAR-1 145 CAR 2 of MUC1-2-A- 219 IRES-hCD19-B VLanti-CD5 46 scFv SIGLEC-15- 146 Construct of MUC1-2- 224 CAR-1B-IRES-hCD19-B VH anti-CD5 47 scFv VL SIGLEC-15- 147 CAR 1 Of MUC1-2-B-212 CAR-1 IRES-hCD19-B VL anti-CD4 48 scFv VH SIGLEC-15- 148 CAR 2 ofMUC1-2-B- 219 CAR-1 IRES-hCD19-B VH anti-CD4 49 VL1 VH1 SIGLEC-15- 149Construct of MUC1- 225 CAR-2 5E5-A-IRES-CD22-A VL anti-CD3 50 VL1 VH2SIGLEC-15- 150 CAR 1 of MUC1-5E5- 202 CAR-3 A-IRES-CD22-A VH anti-CD3 51VL1 VH3 SIGLEC-15- 151 CAR 2 of MUC1-5E5- 226 CAR-4 A-IRES-CD22-A TSHR52 VL1 VH 4 SIGLEC-15- 52 Construct of MUC1- 227 extracellular CAR-55E5-B-IRES-CD22-A domain VH region of 53 VL2 VH 1 SIGLEC-15- 153 CAR 1of MUC1-5E5- 205 BCMA scFv CAR-6 A-IRES-CD22-A VL region of 54 VL2 VH2SIGLEC-15- 154 CAR 2 of MUC1-5E5- 226 BCMA scFv CAR-7 A-IRES-CD22-A VHregion of 55 VL2 VH3 SIGLEC-15- 155 Construct of MUC1- 228 CD14 scFvCAR-8 5E5-A-IRES-CD22-B VL region of CD14 56 VL2 VH4 SIGLEC-15- 156MUC1-5E5-A-IRES- 202 scFv CAR-9 CD22-B CAR 1 VH region of 57 VL1SIGLEC-15-CAR 157 MUC1-5E5-A-IRES- 229 CD33 scFv CD22-B CAR 2 VL regionof CD33 58 VL2 SIGLEC-15-CAR 158 MUC1-5E5-B-IRES- 230 scFv CD22-BCD22CAR 59 VH1 SIGLEC-15-CAR 159 CAR 1 of MUC1-5E5- 205 B-IRES-CD22-BBCMACAR 60 VH2 SIGLEC-15-CAR 160 CAR 2 of MUC1-5E5- 229 B-IRES-CD22-BMUC1CAR 61 VH3 SIGLEC-15-CAR 161 Construct of MUC1-2- 231 A-IRES-CD22-Am19CAR-IRES- 62 VH4 SIGLEC-15-CAR 162 CAR 1 of MUC1-2-A- 210 MUC1CARIRES-CD22-A hCD19CAR-IRES- 63 MUC16-CAR-1 163 CAR 2 of MUC1-2-A- 226MUC1CAR IRES-CD22-A hCD22CAR-IRES- 64 scFv MUC16-CAR-1 164MUC1-2-B-IRES- 232 MUC1CAR CD22-A BCMACAR-IRES- 65 scFv VL MUC16- 165MUC1-2-B-IRES- 212 MUC1CAR CAR-1 CD22-A CAR 1 mCD19CAR-2A- 66 scFv VHMUC16- 166 MUC1-2-B-IRES- 226 MUC1CAR CAR-1 CD22-A CAR 2 hCD19CAR-2A- 67MUC16-CAR-2 167 MUC1-2-A-IRES- 233 MUC1CAR CD22-B hCD22CAR-2A- 68 scFvMUC16-CAR-2 168 MUC1-2-A-IRES- 210 MUC1CAR CD22-B CAR 1 BCMA-2A- 69 scFvVL MUC16- 169 MUC1-2-A-IRES- 229 MUC1CAR CAR-2 CD22-B CAR 2 Tumorassociated 70 scFv VH MUC16- 170 Construct of MUC1-2- 234 MUC1 scFv 1CAR-2 B-IRES-CD22-B Tumor associated 71 KISS1R-CAR 171 CAR 1 ofMUC1-2-B- 212 MUC1 scFv-1 VH IRES-CD22-B Tumor associated 72 Ligentpeptide 172 CAR 2 of MUC1-2-B- 229 MUC1 scFv-1 VL KISS1R-CAR IRES-CD22-BTumor associated 73 ZFLm1 (left) RS aa 173 Construct of MUC1- 235 MUC1scFv-1 VL 5E5-A-IRES-CD14-A CDR 1 L2D8-2 (hCAR 74 ZFLm1 (left) F1 174CAR 1 of MUC1-5E5- 202 VL) A-IRES-CD14-A Tumor associated 75 ZFLm1(left) F2 174 CAR 2 of MUC1-5E5- 236 MUC1 scFv-1 VL A-IRES-CD14-A CDR 3Tumor associated 76 ZFLm1 (left) F3 176 Construct of MUC1- 237 MUC1scFv-1 VH 5E5-B-IRES-CD14-A CDR 1 Tumor associated 77 ZFLm1 (left) F4177 CAR 1 of MUC1-5E5- 205 MUC1 scFv-1 VH B-IRES-CD14-A CDR 2 Tumorassociated 78 ZFLm1 (left) F5 178 CAR 2 of MUC1-5E5- 236 MUC1 scFv-1 VHB-IRES-CD14-A CDR 3 Tumor associated 79 ZFLm1 (left) F6 179 Construct ofMUC1- 238 MUC1 scFv2 5E5-A-IRES-CD14-B Tumor associated 80 ZFRm1-4(right) RS 180 CAR 1 of MUC1-5E5- 202 MUC1 scFv2 VH aa A-IRES-CD14-BTumor associated 81 ZFRm1-4 (right) F1 181 CAR 2 of MUC1-5E5- 239 MUC1scFv2 VL A-IRES-CD14-B Tumor associated 82 ZFRm1-4 (right) F2 182Construct of MUC1-2- 240 MUC1 scFv-2 VL A-IRES-CD14-A CDR 1 Tumorassociated 83 ZFRm1-4 (right) F3 184 CAR 1 of MUC1-2-A- 210 MUC1 scFv-2VL IRES-CD14-A CDR 2 Tumor associated 84 ZFRm1-4 (right) F4 184 CAR 2 ofMUC1-2-A- 236 MUC1 scFv-2 VL IRES-CD14-A CDR 3 {grave over ( )}Tumorassociated 85 δ chain-1 of 185 Construct of MUC1-2- 241 MUC1 scFv-2VHVγ9Vδ2 B-IRES-CD14-A CDR 1 Tumor associated 86 γ chain-2 of Vγ9Vδ2 186CAR 1 of MUC1-2-B- 212 MUC1 scFv-2 VH IRES-CD14-A CDR 2 Tumor associated87 δ chain-2 of Vγ9Vδ2 187 CAR 2 of MUC1-2-B- 236 MUC1 scFv-2 VHIRES-CD14-A CDR 3 GSTA motif 88 Vγ9Vδ2 TCR-1: DG. 188 Construct ofMUC1-2- 242 SF13 γ chain A-IRES-CD14-B Modified PD-1 89 Vγ9Vδ2 TCR-1:DG. 189 CAR 1 of MUC1-2-A- 210 intracellular SF13 δ chain IRES-CD14-Bdomain -1 Modified PD-1 90 Vγ9Vδ2 TCR-2: DG. 190 CAR 2 of MUC1-2-A- 239intracellular SF68: γ chain IRES-CD14-B domain -2 Modified PD-1 91Vγ9Vδ2 TCR-2: DG. 191 Construct of MUC1-2- 243 intracellular SF68: δchain B-IRES-CD14-B domain -3 Modified PD-1 92 Vγ9Vδ2 TCR-3: 192 CAR 1of MUC1-2-B- 212 intracellular 12G12: γ chain IRES-CD14-B domain -4Modified PD-1 93 Vγ9Vδ2 TCR-3: 193 CAR 2 of MUC1-2-B- 239 intracellular12G12: δ chain IRES-CD14-B domain -5 Removed PD-1 94 Vγ9Vδ2 TCR-4: 194Construct of MUC1- 244 intracellular CP.1.15 γ chain 5E5-A-IRES-BCMA-Adomain -1 Removed PD-1 95 TCR-4: CP.1.15δ 195 CAR 1 of MUC1-5E5- 202intracellular chain A-IRES-BCMA-A domain -2 Fokl WC 96 WT CD3-zeta 196CAR 2 of MUC1-5E5- 245 A-IRES-BCMA-A M Fokl 97 Invariant sequence for197 Construct of MUC1- 246 iNKT α chain (hVα24- 5E5-B-IRES-BCMA-AJαQ-TRAC) M Fokl 98 An example for iNKT 198 CAR 1 of MUC1-5E5- 205 βchain sequence B-IRES-BCMA-A (containing Vβ11): γ chain-1 of 99Invariant sequence for 199 CAR 2 of MUC1-5E5- 245 Vy9Vδ2 MAIT α chainB-IRES-BCMA-A ( hAV7S2-AJ33 α chain) (version1) VL anti-CD4-2 100 VHanti- CD4-2 200 Construct of MUC1- 247 5E5-A-IRES-BCMA-B CAR 1 ofMUC1-2- 210 CAR 1 of MUC1-5E5- 205 CAR 1 of MUC1-5E5- 202 A-IRES-CD33-AB-IRES-CD33-A A-IRES-BCMA-B CAR 2 of MUC1-2- 255 CAR 2 of MUC1-5E5- 255CAR 2 of MUC1-5E5- 248 A-IRES-CD33-A B-IRES-CD33-A A-IRES-BCMA-BConstruct 261 Construct ofMUCI- 257 Construct of MUC1- 249 ofMUC1-2-B-5E5-A-IRES-CD33-B 5E5-B-IRES-BCMA-B IRES-CD33-A CAR 1 of MUC1-2- 212 CAR1 of MUC1-5E5- 202 CAR 1 of MUC1-5E5- B-IRES-CD33-A A-IRES-CD33-BB-IRES-BCMA-B CAR 2 of MUC1-2- 255 CAR 2 of MUC1-5E5- 258 CAR 2 ofMUC1-5E5- B-IRES-CD33-A A-IRES-CD33-B B-IRES-BCMA-B Construct 262Construct ofMUCI- 259 Construct of MUC1-2- 250 ofMUC1-2-A-5E5-B-IRES-CD33-B A-IRES-BCMA-A IRES-CD33-B CAR 1 of MUC1-2- 210 CAR 1of MUC1-5E5- 205 CAR 1 of MUC1-2-A- 210 A-IRES-CD33-B B-IRES-CD33-BIRES-BCMA-A CAR 2 of MUC1-2- 258 CAR 2 of MUC1-5E5- 258 CAR 2 ofMUC1-2-A- 245 A-IRES-CD33-B B-IRES-CD33-B IRES-BCMA-A Construct 263Construct ofMUC1-2- 260 Construct of MUC1-2- 251 ofMUC1-2-B-A-IRES-CD33-A B-IRES-BCMA-A IRES-CD33-B CAR 1 of MUC1-2- 212 ConstructofMUC1-2- 253 CAR 1 of MUC1-2-B- 212 B-IRES-CD33-B B-IRES-BCMA-BIRES-BCMA-A CAR 2 of MUC1-2- 258 CAR 1 of MUC1-2-B- 212 CAR 2 ofMUC1-2-B- 245 B-IRES-CD33-B IRES-BCMA-B IRES-BCMA-A Construct 254MUC1-2-B-IRES- 248 Construct of MUC1-2- 252 ofMUC1-5E5-A- BCMA-B CAR 2A-IRES-BCMA-B IRES-CD33-A CAR 1 of MUC1- 202 MUC1-5E5-B-IRES- 256 CAR 1of MUC1-2-A- 210 5E5-A-IRES- CD33-A IRES-BCMA-B CD33-A CAR 2 of MUC1-255 CAR 2 of MUC1-2-A- 248 MUC1-5e5Panko- 264 5E5-A-IRES- IRES-BCMA-Benhanced scFc CD33-A MUC1-Panko5e5 - 265 hinge and/or 266 hinge and/or267 enhanced scFc transmembrane transmembrane domain A domain B hingeand/or 268 hinge and/or 269 MUC1-5e5Panko- 270 transmembranetransmembrane enhanced scFc domain C domain D A 41BB CD2 zetaMUC1-5e5Panko- 271 MUC1-5e5Panko- 272 MUC1-5e5Panko- 273 enhancedenhanced scFc enhanced scFc ScFc B 41BB CD2 C 41BB CD2 zeta D 41BB CD2zeta zeta MUC1-Panko5e5- 274 MUC1-Panko5e5- 275 MUC1-Panko5e5- 276enhanced enhanced scFc enhanced scFc C scFc A 41BB CD2 B 41BB CD2 zeta41BB CD2 zeta zeta MUC1-Panko5e5- 277 GS linker 278 Construct of TSHR279 enhanced CAR ScFc D 41BB CD2 zeta CD8a Hinge & 280 IL-17C 296 IL12-IgG4 Hinge 313 transmembrane Nucleic acid &CD8a Sequence transmembraneCD8a 281 IL-17C 297 IL12Rβ2 cytoplasmic 314 transmembrane aa SequenceIgG4 Hinge&CD8a 282 IL-17D 298 IL18R1 cytoplasmic 315 transmembraneNucleic acid Sequence IL-2 283 IL-17D 299 IL23R cytoplasmic 316 Nucleicacid aa Sequence Sequence IL-2 285 IL-17F 300 Gp130 (IL6ST) 317 aaSequence Nucleic acid cytoplasmic Sequence IL-6 286 IL-17F 301 IL15Ra,cytoplasmic 318 Nucleic acid aa Sequence Sequence IL-6 287 IL-23A 302IL12Rβ1 319 aa Sequence Nucleic acid cytoplasmic Sequence IL-7 288IL-23A 303 41BB + cd3zeta + IL 320 Nucleic acid aa Sequence receptorcytoplasmic Sequence region IL-7 289 IL-18 304 scFv + hinge + 321 aaSequence Nucleic acid transmembrane + Sequence 41BB + IL receptorcytoplasmic region + cd3zeta IL-15 290 IL-18 305 scFv + hinge + 322Nucleic acid aa Sequence transmembrane + Sequence IL receptorcytoplasmic region + 41BB + cd3zeta IL-15 291 IL-12 αp35 306 41BBpromoter 323 aa Sequence Nucleic acid Sequence IL-17A 292 IL-12αp35 307CD25 enhancer + 324 Nucleic acid aa Sequence minimal TK promoterSequence IL-17A 293 IL-12βp40 308 CD69 enhancer + 325 aa SequenceNucleic acid minimal TK promoter Sequence IL-17B 294 IL-12 βp40 309IFN-gamma promoter 326 Nucleic acid aa Sequence Sequence IL-17B 295Fusion IL-12 310 2A 327 aa Sequence Hypoxia promoter 330 Fusion IL-23311 IFN-γ 328 (example 2) Transcription factor binding sites. Hypoxiapromoter 331 IL12- CD8a Hinge & 312 hPDE5 329 (example 3) transmembraneTranscription factor binding sites. NFAT promoter 332 Hypoxia promoter338 TNF-alpha 347 (example 1) (example 1) the Transcription minimalpromoter factor binding sites. NFAT promoter 333 Hypoxia promoter 339Lymphotoxin 348 (example 2) (example 2) the beta (TNF-C) Transcriptionminimal promoter factor binding sites. FOXP3 promoter 334 Hypoxiapromoter 340 OX40L 349 (example1) (example 3) the Transcription minimalpromoter factor binding sites. Hypoxia promoter 335 NFAT promoter 341CD154 350 (example 1) (example 1) the Transcription minimal promoterfactor binding sites. NFkB promoter 336 NFAT promoter 342 FasL 351(example1) (example 2) the Transcription minimal promoter factor bindingsites. NFkB promoter 337 FOXP3 promoter 343 CD70 352 (example2)(example1) the Transcription minimal promoter factor binding sites.CXCI1 364 FOXP3 promoter 344 CD153 353 (example2) the minimal promoterCXCL2 365 NFkB promoter 345 4-1BB ligand 354 (example1) the minimalpromoter CXCL3 366 NFkB promoter 346 TRAIL 355 (example2) the minimalpromoter CXCL4 367 CXCL11 374 RANKL 356 CXCL5 368 CXCL12 375 TWEAK 357CXCL6 369 CXCL13 376 APRIL 358 CXCL7 370 CXCL14 377 LIGHT 359 CXCL8 371CCL1 378 NGF, 360 CXCL9 372 CCL2 379 TNFSF18 361 CXCL10 373 CCL3 380TNFSF15 362 ICOS ligand 405 CCI3L1 381 Ectodysplasin-A 363 CD80 406 CCI4382 IL2 receptor CD 415 CD86 407 CCI5 383 IL6 receptor CD 418 scFvagainst PD1 408 CCI7 384 IL7 receptor CD 421 scFv against PDL1 409 CCI8385 IL12 receptor CD 424 B7-H3 scFv 1 410 CCI11 386 IL15 receptor CD 427B7-H3 scFv2 411 CCL13 387 IL21 receptor CD 430 B7-H3 scFv3 412 CCI14 388IL23 receptor CD 433 IL2 receptor ED 413 CCL15 389 CD4 TM 434 IL6receptor ED 416 CCI16 390 CD8 TM 436 IL7 receptor ED 419 CCL17 391 CD27TM 438 IL12 receptor ED 422 CCI18 392 CD28 TM 440 IL15 receptor ED 425CCL19 393 CD137 TM 442 IL21 receptor ED 428 CCL20 394 PD1 TM 444 IL23receptor ED 431 CCL21 395 PDL1 TM 446 IL2 receptor TM 414 CCL22 396 CD4CD 435 IL6 receptor TM 417 CCL23 397 CD8 CD 437 IL7 receptor TM 420CCL24 398 CD27 CD 439 IL12 receptor TM 423 CCL25 399 CD28 CD 441 IL15receptor TM 426 CCL26 400 CD137 CD 443 IL21 receptor TM 429 CCL27 401PD1 CD 445 IL23 receptor TM 432 CCL28 402 PDL1 CD 447 IL2 448Lymphotactin 403 IL21 452 IL7 449 CX3CL1 404 IL23 453 IL12 450 HifVHL-interaction 457 IL33 454 domain: Hif amino acid 344-417 IL15 451 Hifamino acid 380- 458 TNFα 455 603 siglec-15 antigen 460 siglec-15 antigen1 459 IFNγ point mutation 456 2 siglec-15 antigen 461 siglec-15 antigen6 464 GS linker sequence 467 3 siglec-15 antigen 462 siglec-15 antigen 7465 EA linker sequence 468 4 siglec-15 antigen 463 siglec-15 antigen 8466 NFAT6x + minimal 469 5 IL12 promoter TM: Transmembrane domain CD:cytoplasmic domain EM: Extracellular daemon

TABLE 3 transcription Expression Conditions factors or notes Example ofconstructs Hif1a hypoxia-induced Hif1a binding site + minimal expressionpromoter + CDS of IL NFAT transcription factor in NFAT binding site +minimal immune response promoter + CDS of IL FOXP3 transcription factorin FOXP3 binding site + minimal T-reg promoter + CDS of IL NFkBtranscription factor in NFkB binding site + minimal immune responsepromoter + CDS of IL

TABLE 4 isolated nucleic acid sequence Nucleic Acid Construct usingEncoded Peptides 1 CAR + P2A + IL + CD8a Hinge & transmembrane 2 CAR +P2A + IL + IgG4 Hinge & CH2CH3 & CD4 transmembrane 3 CAR +Hypoixa/NFAT/FOXP3/NFkB promoter + IL + CD8a Hinge & transmembrane 4CAR + Hypoixa/NFAT/FOXP3/NFkB promoter + IL + CD8a IgG4Hinge & CH2CH3 &CD4 transmembrane 5 scFv + hinge + transmembrane + 41BB + cd3zeta + ILreceptor cytoplasmic region 6 scFv + hinge + transmembrane + 41BB + ILreceptor cytoplasmic region + cd3zeta 7 scFv + hinge + transmembrane +IL receptor cytoplasmic region + 41BB + cd3zeta

Exemplary Embodiments

The following are exemplary embodiments:

1. An isolated nucleic acid sequence comprising a first nucleic acidsequence and a second or an additional nucleic acid sequence, the firstnucleic acid sequence encoding a chimeric antigen receptor (CAR), thesecond or additional nucleic acid sequence encoding one or moretherapeutic agents. For example, the one or more therapeutic agents areor comprise of IL-2, IL-6, IL-7, IL-15, IL-17, IL-23, or a combinationthereof.2. An isolated nucleic acid sequence comprising a first nucleic acidsequence and a second or an additional nucleic acid sequence, the firstnucleic acid sequence encoding a chimeric antigen receptor (CAR), thesecond or additional nucleic acid sequence encoding a therapeutic agentthat is or comprises at least one of TNFRSF superfamily member receptoractivation antibodies or membrane-bound forms thereof, TNFRSFsuperfamily member ligands or the membrane-bound form thereof,chemokines or membrane-bound forms thereof, antibodies to thechemokines, or antibodies to receptors of the chemokines or themembrane-bound forms thereof, and D28 family's ligands that correspondto the sequences in Table 2-4.3. A population of CAR cells comprising the first nucleic acid sequenceand the additional nucleic acid sequence of embodiments 1 or 2, whereinthe CAR cells comprise lymphocyte, leukocyte, or PBMC.4. The population of CAR cells of embodiment 3, wherein the CAR and theone or more therapeutic agents are produced in the form of apolyprotein, which is cleaved to generate separate CAR and therapeuticagent molecules.5. The population of CAR cells of embodiment 4, wherein the polyproteincomprises a cleavable moiety between the CAR and the therapeutic agent,the cleavable moiety comprises a 2A peptide, the 2A peptide comprisesP2A or T2A, and/or the CAR and the therapeutic agent are eachconstitutively expressed.6. The population of CAR cells of embodiment 3, wherein the CAR cellscomprise:a third nucleic acid sequence encoding a second or an additional CARbinding an antigen that is different from the CAR, or the second oradditional CAR binding a solid tumor antigen, and the CAR binds anantigen of a white blood cell.7. A pharmaceutical composition comprising the population of CAR cellsof one of embodiments 3-6. The pharmaceutical composition is used totreat a patient having a solid tumor.8. A method of inducing or causing a T cell response in a subject inneed thereof and/or treating a tumor of the subject, the methodcomprising administering an effective amount of the composition ofembodiment 7 to the subject.9. A modified cell comprising one or more CARs, wherein the cell isengineered to express and secrete a therapeutic agent that is orcomprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23.10. A method of inducing or causing or enhancing T cell response,treating cancer, or enhancing cancer treatment, the method comprising:administrating an effective amount of the composition of T cellscomprising one or more CARs, wherein the cell is engineered to expressand secrete one or more therapeutic agents. For example, the therapeuticagent is or comprises IL-2, IL-6, IL-7, IL-15, IL-17, IL-23, or acombination thereof and the T cell response is enhanced as compared tothe administration of T cells that do not express or secrete thetherapeutic agent.11. A method of inducing or causing or enhancing T cell response,treating cancer, or enhancing cancer treatment, the method comprising:administering an effective amount of the composition of a population ofT cells comprising a CAR; and administering an effective amount of oneor more therapeutic agents. For example, the therapeutic agent is orcomprises IL-2, IL-6, IL-7, IL-15, IL-17, IL-23, or a combinationthereof, wherein the T cell response is enhanced as compared to theadministration of CAR T cells without the administration of therapeuticagent.12. The method of embodiment 11, wherein administering an effectiveamount of the therapeutic agent comprises intravenous delivery of anamount of human IL-6 in the range of about 0.5-50 ug per kilogram ofbody weight.13. The modified cell or the method of one of embodiments 9-12, whereinthe T cell comprises a second or an additional CAR binding a solid tumorantigen, and the first CAR binds an antigen of a white blood cell.14. The modified cell or the method of embodiment 13, wherein the solidtumor antigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1,GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6,UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2,FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2,EpCAM, EGFRvIII, PSCA, or EGFR, and the B cell antigen is CD19, CD20,CD22, or BCMA.15. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-14, wherein the therapeutic agent is IL-6 orIL-7.16. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-14, wherein the CAR comprises anextracellular domain, a transmembrane domain, and an intracellulardomain, the extracellular domain binding an antigen.17. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 16, wherein the intracellular domain comprises aco-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.18. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 17, wherein the antigen is Epidermal growth factorreceptor (EGFR), Variant III of the epidermal growth factor receptor(EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin(MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonicantigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.19. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-18, wherein the modified cell or T cellscomprise a dominant negative PD-1 mutant such that PD-1/PDI-1 signalingpathway of the cell is interfered.20. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-19, wherein the nucleic acid sequenceencoding the therapeutic agent is present in the modified cell in arecombinant DNA construct, in an mRNA, or in a viral vector.21. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-19, wherein the modified cell comprises anmRNA encoding the therapeutic agent, wherein the mRNA is not integratedinto the genome of the modified cell.22. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-21, wherein the therapeutic agentcorresponds to at least one of sequence listed in Table 2.23. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 2-21, wherein the modified cell comprises anucleic acid sequence comprising a promoter comprising a binding sitefor a transcription modulator that modulates the expression of thetherapeutic agent in the cell.24. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 23, wherein the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB.25. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 24, wherein the promoter is responsive to thetranscription modulator.26. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 25, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives expression of the therapeutic agent in the cell.27. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 23, wherein the promoter and the binding site correspondto the sequences listed in Table 2-4.28. An isolated nucleic acid sequence comprising a first nucleic acidsequence and a second nucleic acid sequence, the first nucleic acidsequence encoding a chimeric antigen receptor (CAR), the second nucleicacid sequence encoding a therapeutic agent and a transmembrane domainsuch that the therapeutic agent is associated or bound to cell membrane.Examples of the isolated nucleic acid sequence are listed in Table 4(1-4)29. A modified cell comprising the isolated nucleic acid sequence ofembodiment 28.30. A pharmaceutical composition comprising the population of the cellsof embodiment 3.31. A method of inducing or causing T cell response in a subject in needthereof and/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 30 tothe subject.32. A modified cell comprising a first nucleic acid sequence encoding aCAR, and a second nucleic acid sequence encoding a therapeutic agent anda transmembrane domain such that the therapeutic agent is associated orbound to the membrane of the modified cell.33. The modified cell of one of embodiments 29-32, wherein thetherapeutic agent is a cytokine.34. The modified cell of embodiment 33, wherein the cytokine comprisesmultiple submits, the second nucleic acid encodes the multiple subunits,one or more linkers connecting the multiple subunits, and thetransmembrane domain.35. The modified cell of embodiment 33, wherein the second nucleic acidsequence comprises a nucleic acid sequence of or encodes an amino acidsequence of SEQ ID NO: 280-313.36. The modified cell of embodiment 33, wherein the cytokine is orcomprises at least one of IL-2, IL-6, IL-7, IL-12, IL-15, IL-17, IL-18,and IL-23.37. The modified cell of any one of embodiments 29-36, wherein the CARcomprises an extracellular domain, a transmembrane domain, and anintracellular domain, wherein the extracellular domain binds an antigen.38. The modified cell of embodiment 37, wherein the intracellular domaincomprises a co-stimulatory domain that comprises an intracellular domainof a co-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.39. The modified cell of embodiment 37, wherein the antigen is Epidermalgrowth factor receptor (EGFR), Variant III of the epidermal growthfactor receptor (EGFRvIII), Human epidermal growth factor receptor 2(HER2), Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA),Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX(CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125),Cluster of differentiation 133 (CD133), Fibroblast activation protein(FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folatereceptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3,CD5, B-Cell Maturation Antigen (BCMA), or CD4.40. The modified cell of one of embodiments 29-39, wherein the secondnucleic acid sequence comprises a promoter comprising a binding site fora transcription modulator that modulates the expression of thetherapeutic agent in the cell.41. The modified cell of embodiment 40, wherein the transcriptionmodulator is or includes Hif1a, NFAT, FOXP3, and/or NFkB.42. A method of inducing or causing or enhancing T cell response,treating cancer, or enhancing cancer treatment, the method comprising:administrating an effective amount of the composition of the modifiedcells of one of embodiments 29-41.43. An isolated nucleic acid sequence encoding a binding moleculecomprising an extracellular domain, a transmembrane domain, and anintracellular domain, the extracellular domain binds an antigen, theintracellular domain comprising a cytoplasmic domain of a receptor of atherapeutic agent. Examples of the isolated nucleic acid sequence arelisted in Table 4 (5-8)44. A cell comprising the isolated nucleic acid sequence of embodiment43.45. A pharmaceutical composition comprising the population of the cellsof embodiment 44.46. A method of inducing or causing T cell response in a subject in needthereof and/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 45 tothe subject.47. A modified cell comprising a binding molecule comprising anextracellular domain, a transmembrane domain, and an intracellulardomain, the extracellular domain binds an antigen, the intracellulardomain comprising a cytoplasmic domain of a therapeutic agent.48. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-47, wherein the therapeutic agent is a cytokine.49. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-47, wherein the receptor of the therapeutic agent isor comprises IL12Rβ2, IL18R1, IL123R, GP130, IL15Ra, or IL12Rβ1.50. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-47, wherein the therapeutic agent is or comprisesIL12, IL18, IL123, IL-6, or IL-15.51. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-47, wherein the cytoplasmic domain is or comprises atleast one of the amino acid sequences of SEQ ID Nos: 314-319.52. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-47, wherein the modified cell comprises the isolatednucleic acid sequence comprising any one of the amino acid sequences ofSEQ ID Nos: 320-322.53. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-51, wherein the modified cell comprises an additionalnucleic acid sequence, the isolated nucleic acid sequence comprisesadditional nucleic acid sequence, and the additional nucleic acidsequence comprises 41-BB domain and CD3 Zeta domain, a nucleic acidsequence encoding the cytoplasmic domain is located between the 41-BBdomain and CD3 zeta domain.54. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-51, wherein the modified cell comprises an additionalnucleic acid sequence, the isolated nucleic acid sequence comprisesadditional nucleic acid sequence, and the additional nucleic acidsequence comprises 41-BB domain and CD3 Zeta domain, a nucleic acidsequence encoding the cytoplasmic domain is located before the 41-BBdomain ordered from a N-terminal of the cytoplasmic domain.55. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-51, wherein the modified cell comprises an additionalnucleic acid sequence, the isolated nucleic acid sequence comprisesadditional nucleic acid sequence, and the additional nucleic acidsequence comprises 41-BB domain and CD3 Zeta domain, a nucleic acidsequence encoding the cytoplasmic domain is located after the CD3 Zetadomain ordered from a N-terminal of the cytoplasmic domain.56. The isolated nucleic acid sequence and the modified cell of any oneof embodiments 43-55, wherein the intracellular domain comprises aco-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.57. The isolated nucleic acid sequence and the modified cell ofembodiment 56, wherein the antigen is Epidermal growth factor receptor(EGFR), Variant III of the epidermal growth factor receptor (EGFRvIII),Human epidermal growth factor receptor 2 (HER2), Mesothelin (MSLN),Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen(CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.58. The modified cell or the cell of any one of embodiments 44-57,wherein a signaling pathway is activated when the binding molecule bindsto the antigen.59. An isolated nucleic acid sequence comprising a nucleic acid sequenceand an additional nucleic acid sequence, the nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the additional nucleic acidsequence encoding a therapeutic agent that is or comprises at least oneof IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23.60. An isolated nucleic acid sequence comprising a nucleic acid sequenceand an additional nucleic acid sequence, the nucleic acid sequenceencoding a chimeric antigen receptor (CAR), the additional nucleic acidsequence encoding a therapeutic agent that is or comprises at least oneof TNFRSF superfamily member receptor activation antibodies ormembrane-bound forms thereof, TNFRSF superfamily member ligands or themembrane-bound form thereof, chemokines or membrane-bound forms thereof,antibodies to the chemokines, or antibodies to receptors of thechemokines or the membrane-bound forms thereof, and D28 family's ligandsthat correspond to the sequences in Table 2-4.61. A population of CAR cells comprising the nucleic acid sequence andthe additional nucleic acid sequence of embodiments 59 or 60, whereinthe CAR cells comprise lymphocyte, leukocyte, or PBMC.62. The population of CAR cells of embodiment 61, wherein the CAR andthe therapeutic agent are produced in the form of a polyprotein, whichis cleaved to generate separate CAR and therapeutic agent molecules.63. The population of CAR cells of embodiment 62, wherein thepolyprotein comprises a cleavable moiety between the CAR and thetherapeutic agent, the cleavable moiety comprises a 2A peptide, the 2Apeptide comprises P2A or T2A, and/or the CAR and the therapeutic agentare each constitutively expressed.64. The population of CAR cells of embodiment 61, wherein the CAR cellscomprise: a third nucleic acid sequence encoding an additional CARbinding to an antigen that is different from the CAR, orthe additional CAR binding a solid tumor antigen, and the CAR binds anantigen of a white blood cell.65. A pharmaceutical composition comprising the population of the CARcells of one of embodiments 61-64.66. A method of causing T cell response in a subject in need thereofand/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 65 tothe subject.67. A modified cell comprises one or more CARs, wherein the cell isengineered to express and secrete a therapeutic agent that is orcomprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23.68. A method of causing or enhancing T cell response, treating cancer,or enhancing cancer treatment, the method comprising: administrating aneffective amount of the composition of T cells comprising one or moreCARs, wherein the cell is engineered to express and secrete atherapeutic agent that is or comprises at least one of IL-2, IL-6, IL-7,IL-15, IL-17, and IL-23, and the T cell response is enhanced as comparedto the administration of T cells that do not express or secrete thetherapeutic agent.69. A method of causing or enhancing T cell response, treating cancer,or enhancing cancer treatment, the method comprising:administering an effective amount of the composition of a population ofT cells comprising a CAR; andadministering an effective amount of a therapeutic agent that is orcomprises at least one of IL-2, IL-6, IL-7, IL-15, IL-17, and IL-23,wherein the T cell response is enhanced as compared to theadministration of CAR T cells without the administration of therapeuticagent.70. The method of embodiment 69, wherein the administering the effectiveamount of the therapeutic agent comprises intravenous delivery of anamount of human IL-6 in the range of about 0.5-50 ug per kilogram ofbody weight.71. The modified cell or the method of one of embodiments 67-70, whereinthe T cell comprises an additional CAR binding a solid tumor antigen,and the CAR binds an antigen of a white blood cell.72. The modified cell or the method of embodiment 71, wherein the solidtumor antigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1,GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6,UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2,FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2,EpCAM, EGFRvIII, PSCA, or EGFR, and the B cell antigen is CD19, CD20,CD22, or BCMA.73. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-72, wherein the therapeutic agent is IL-6or IL-7.74. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-72, wherein the CAR comprises anextracellular domain, a transmembrane domain, and an intracellulardomain, the extracellular domain binds an antigen.75. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 74, wherein the intracellular domain comprises aco-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.76. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 75, wherein the antigen is Epidermal growth factorreceptor (EGFR), Variant III of the epidermal growth factor receptor(EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin(MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonicantigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.77. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-76, wherein the modified cell or T cellscomprise a dominant negative PD-1 mutant such that PD-1/PDI-1 signalingpathway of the cell is interfered.78. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-77, wherein the therapeutic agent ispresent in the modified cell in a recombinant DNA construct, in an mRNA,or in a viral vector.79. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-77, wherein the modified cell comprises atherapeutic agent mRNA encoding the therapeutic agent, and the mRNA isnot integrated into the genome of the modified cell.80. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-79, wherein the therapeutic agentcorresponds to at least one of sequence listed in Table 2-4.81. The isolated nucleic acid sequence, the modified cell, or the methodof any one of embodiments 60-79, wherein the modified cell comprises anucleic acid sequence comprising a promoter comprising a binding sitefor a transcription modulator that modulates the expression of thetherapeutic agent in the cell.82. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 81, wherein the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB.83. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 82, wherein the promoter is responsive to thetranscription modulator.84. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 83, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives expression of the therapeutic agent in the cell.85. The isolated nucleic acid sequence, the modified cell, or the methodof embodiment 81, wherein the promoter and the binding site correspondto the sequences listed in Table 2-4.86. A modified cell comprises one or more CARs, wherein the cell isengineered to express and secrete a therapeutic agent such as acytokine.87. A method of causing or enhancing T cell response, treating cancer,or enhancing cancer treatment, the method comprising: administrating aneffective amount of the composition of T cells comprising one or moreCARs, wherein the cell is engineered to express and secrete atherapeutic agent such as a cytokine.88. The modified cell or the method of one of embodiments 86-87, whereinthe therapeutic agent that is or comprises IFN-γ.89. The modified cell or the method of one of embodiments 86-88, whereinthe therapeutic agent that is or comprises IL-6 or IFN-γ, or acombination thereof. For example, the therapeutic agent may comprise SEQID NO: 287 or 328.90. The modified cell or the method of one of embodiments 86-89, whereinthe therapeutic agent that is or comprises IL-15 or IL-12, or acombination thereof.91. The modified cell or the method of one of embodiments 86-90, whereinthe small protein or the therapeutic agent is or comprises a recombinantor native cytokine.92. The modified cell or the method of one of embodiments 86-91, whereinthe therapeutic agent comprises a FC fusion protein associated with asmall protein.93. The modified cell or the method of one of embodiments 86-92, whereinthe small protein is or comprises IL-12, IL-15, IL-6 or IFN-γ.94. The modified cell or the method of one of embodiments 86-93, whereinexpression and/or secretion is regulated by a controlling system such asan inducible expression system, or the modified cell is regulated by aninducible suicide expression.95. The modified cell or the method of one of embodiments 86-94, whereinthe therapeutic agent activates macrophages and/or dendritic cells.96. The modified cell or the method of one of embodiments 86-95, whereinthe therapeutic agent causes macrophages to remove granulocytes.97. The modified cell or the method of one of embodiments 86-96, whereinthe therapeutic agent inhibits or suppresses growth of cancer cells.98. The modified cell or the method of one of embodiments 86-97, whereinthe therapeutic agent is or comprises a recombinant or a native protein.99. The modified cell or the method of one of embodiments 86-98, whereinthe modified cell comprises a modified programmed cell death protein 1(PD-1) that is a dominant negative PD-1.100. The modified cell or the method of one of embodiments 86-99,wherein the one or more CARs comprise a CAR targeting a tumor cell.101. The modified cell or the method of one of embodiments 86-100,wherein the one or more CARs comprise a CAR binding a solid tumorantigen and an additional CAR binding a blood cell antigen such as a Bcell antigen.102. The modified cell or the method of one of embodiments 86-100,wherein the solid tumor antigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C,GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1,SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR,GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP,CEA, EphA2, FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2,FR-α, ErbB2, EpCAM, EGFRvIII, or EGFR, and the B cell antigen is CD19,CD20, CD22, or BCMA.103. The modified cell or the method of one of embodiments 86-102,wherein the solid tumor antigen comprises B7, CAIX, CD123, CD133, CD171,CD171/L1-CAM, CEA, Claudin 18.2, cMet, CS1, CSPG4, Dectin1, EGFR, EGFRvIII, EphA2, ERBB receptors, ErbB T4, ERBB2, FAP, Folate receptor 1,FITC, Folate receptor 1, FSH, GD2, GPC3, HA-1 H/HLA-A2, HER2, IL-11Ra,IL13 receptor a2, IL13R, IL13Rα2 (zetakine), Kappa, Leukemia, LewisY,Mesothelin, MUC1, NKG2D, NY-ESO-1, PSMA, ROR-1, TRAIL-receptor1, orVEGFR2, and the B cell antigen is CD19, CD20, CD22, or BCMA.104. The modified cell or the method of one of embodiments 86-103,wherein the T cell response is enhanced as compared to theadministration of CAR T cells that do not express or secrete thetherapeutic agent.105. The modified cell or the method of one of embodiments 86-104,wherein the modified cell comprises a nucleic acid sequence encodinghTERT or a nucleic acid encoding SV40LT, or a combination thereof,wherein the nucleic acid sequence encoding hTERT or a nucleic acidencoding SV40LT, or a combination thereof is integrated into the genomeof the modified T cell, and the modified T cell constitutively expresseshTERT, SV40LT, or a combination thereof.106. The modified cell or the method of one of embodiments 86-105,wherein expression of the nucleic acid sequence encoding hTERT, anucleic acid encoding SV40LT, or a combination thereof, is regulated byan inducible expression system, and/or the modified T cell comprises anucleic acid sequence encoding a suicide gene.107. The modified cell or the method of one of embodiments 86-106,wherein the modified cell is derived from a healthy donor or thesubject.108. The modified cell or the method of one of embodiments 86-107,wherein the TRAC gene of the modified cell is inactivated.109. The modified cell or the method of one of embodiments 86-108,wherein the modified cell has a reduced graft-versus-host disease (GVHD)response in a bioincompatible human recipient as compared to the GVHDresponse of the primary human T cell in response to allogenic CAR Ttreatment.110. The modified cell or the method of one of embodiments 86-109,wherein the modified cell has reduced amount PD-1 or has a dominantnegative PD-1 such that an signaling pathway of the PD-1 is blocked.111. The modified cell or the method of one of embodiments 86-110,wherein the modified cell has reduced amount PD-1 or has a dominantnegative PD-1 such that an signaling pathway of the PD-1 is blocked, thetherapeutic agent is IL-12 or IFN-γ, or a combination thereof.112. The modified cell or the method of an embodiments 86-111, whereinthe modified cell has reduced amount PD-1 or has a dominant negativePD-1 such that an signaling pathway of the PD-1 is blocked, thetherapeutic agent comprises a CD40 agonist such as CP-870,893 fromPfizer.113. The modified cell or the method of an embodiments 86-112, whereinthe modified cell comprises an additional therapeutic agent, themodified cell comprises a nucleic acid sequence encoding the therapeuticagent and an additional nucleic acid sequence encoding the additionaltherapeutic agent, and the nucleic acid sequence and the additionalnucleic acid sequence are connected by an IRES element or a thirdnucleic acid sequence encoding a 2A peptide.114. The modified cell or the method of embodiment 113, wherein thetherapeutic agent is IL-6, and the additional therapeutic agent isIFN-γ.115. The modified cell or the method of embodiment 113, wherein thetherapeutic agent is IL-12, and the additional therapeutic agent isIFN-γ.116. The modified cell or the method of claim 113, wherein thetherapeutic agent is CD40, and the additional therapeutic agent isIFN-γ.117. The modified cell or the method of an embodiments 86-116, whereinthe modified cell comprises a nucleic acid sequence comprising apromoter comprising a binding site for a transcription modulator thatmodulates the expression and/or secretion of the therapeutic agent inthe cell.118. The modified cell or the method of any one of embodiments 86-116,wherein the transcription modulator is or includes Hif1a, NFAT, FOXP3,and/or NFkB.119. The modified cell or the method of embodiment 118, wherein thepromoter is responsive to the transcription modulator.120. The modified cell or the method of any one of embodiments 118-119,wherein the promoter is operably linked to the nucleic acid sequenceencoding the therapeutic agent such that the promoter drives expressionand/or secretion of the therapeutic agent in the cell.121. The modified cell or the method of any one of embodiments 118-120,wherein the promoter comprises at least one of SEQ ID Nos: 323-325.122. The modified cell or the method of any one of embodiments 86-120,wherein the modified cell comprises one or more nucleic acid sequencesencoding a stimulus response element and encoding one or more CARsand/or the therapeutic agent, and the stimulus response elementcomprises at least one portion of the cGMP-specific 3′,5′-cyclicphosphodiesterase or a molecule derived of, for example, SEQ ID NO: 329.123. The modified cell or the method of embodiment 122, whereinexpression of the one or more CARs and/or the therapeutic agent isligand dependent.124. The modified cell or the method of embodiment 122, whereinexpressed the one or more CARs and/or the therapeutic agent aredestabilized or degraded in the absence of a corresponding ligand.125. The modified cell or the method of any one of embodiments 122-124,wherein modified cell comprises one or more nucleic acid sequencesencoding at least one portion of the cGMP-specific 3′,5′-cyclicphosphodiesterase or a molecule derived of, for example, SEQ ID NO: 329appended to or associated with the therapeutic agent such thatexpression of the therapeutic agent is ligand dependent, and thetherapeutic agent is or comprises IL6 or IFN-γ, or a combinationthereof.126. A fusion protein comprising a scFv binding PD-1 or PDL1, a linker,an extracellular domain, a transmembrane domain, and a cytoplasmicdomain, wherein the transmembrane domain is selected from a groupconsist of a transmembrane domain of a receptor of IL15, IL2, IL7, IL6,IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, IFNγ, and IFN as wellas siglec-15 antigen, and the cytoplasmic domain is selected from agroup consist of a cytoplasmic domain of receptor of the receptor ofIL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα,IFNγ, and IFNβ as well as siglec-15 antigen, and the extracellulardomain is selected from a group consist of an extracellular domain ofthe receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33,TNFα, TNFβ, IFNα, IFNγ, and IFNβ as well as siglec-15 antigen.127. A fusion protein comprising a scFv binding PD-1 or PDL1, a linker,a transmembrane domain, and a cytoplasmic domain, wherein thetransmembrane domain is selected from a group consist of a transmembranedomain of a receptor of CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1as well as siglec-15 antigen, and the cytoplasmic domain is selectedfrom a group consist of a cytoplasmic domain of receptor of the receptorof CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1 as well as siglec-15antigen.128. A fusion protein comprising a cytokine is selected from a groupconsist of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα,TNFβ, IFNα, and IFNβ, a linker, an extracellular domain, a transmembranedomain, and a cytoplasmic domain, wherein the transmembrane domain isselected from a group consist of a transmembrane domain of the receptorof IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα,IFNγ, and IFNβ as well as siglec-15 antigen, and the cytoplasmic domainis selected from a group consist of a cytoplasmic domain of receptor ofthe receptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33,TNFα, TNFβ, IFNα, and IFNβ, and the extracellular domain is selectedfrom a group consist of an extracellular domain of the receptor of IL15,IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, andIFNβ.129. A fusion protein comprising a cytokine is selected from a groupconsist of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα,TNFβ, IFNα, and IFNβ, a linker, a transmembrane domain, and acytoplasmic domain, wherein the transmembrane domain is selected from agroup consist of a transmembrane domain of a receptor of CD4, CD8, CD28,CD27, CD25, CD137, PD1 and PDL1 as well as siglec-15 antigen, and thecytoplasmic domain is selected from a group consist of a cytoplasmicdomain of receptor of the receptor of CD4, CD8, CD28, CD27, CD25, CD137,PD1 and PDL1 as well as siglec-15 antigen.130. A fusion protein comprising a binding domain binding a ligand or areceptor of an immune checkpoint molecule and a docking molecule,wherein the immune checkpoint molecule is selected from the groupconsisting of programmed death 1 (PD-1), cytotoxic T lymphocyteantigen-4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T cellimmunoglobulin mucin-3 (TIM-3), lymphocyte-activation protein 3 (LAG-3),T cell immunoreceptor with Ig and ITIM domains (TIGIT),leukocyte-associated immunoglobulin-like receptor 1 (LAIRI), naturalkiller cell receptor 2B4 (2B4), VISTA (its receptor), and CD 160, andthe docking molecule associates the binding domain with a cell.131. The fusion protein of embodiment 130, wherein the docking moleculecomprises a linker, a transmembrane domain, and a cytoplasmic domain.132. The fusion protein of embodiment 131, wherein the transmembranedomain is selected from a group consist of a transmembrane domain of areceptor of IL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα,TNFβ, IFNα, and IFNβ, and the cytoplasmic domain is selected from agroup consist of a cytoplasmic domain of receptor of the receptor ofIL15, IL2, IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα,and INFβ.133. The fusion protein of embodiment 131, wherein the docking moleculefurther comprises an extracellular domain. 134. The fusion protein ofembodiment 133, wherein the extracellular domain is selected from agroup consist of an extracellular domain of the receptor of IL15, IL2,IL7, IL6, IL12, IL18, IL21, IL23, IL 33, TNFα, TNFβ, IFNα, and INFβ.135. The fusion protein of embodiment 130, wherein the docking moleculecomprises a linker, a transmembrane domain, and a cytoplasmic domain.136. The fusion protein of embodiment 135, wherein the transmembranedomain is selected from a group consist of a transmembrane domain of areceptor of CD4, CD8, CD28, CD27, CD25, CD137, PD1 and PDL1 as well assiglec-15 antigen, and the cytoplasmic domain is selected from a groupconsist of a cytoplasmic domain of receptor of the receptor of CD4, CD8,CD28, CD27, CD25, CD137, PD1 and PDL1 as well as siglec-15 antigen.137. The fusion protein of one of embodiments 130-136, wherein thebinding domain is a scFv.138. The fusion protein of embodiment 130, wherein the binding domain isa scFv binding CD80 or CD86.139. The fusion protein of embodiment 138, wherein the docking moleculecomprises or is wide type or modified CTLA4 or PD-1.140. The fusion protein of embodiment 130, wherein the binding domain isa scFv binding VISTA.141. The fusion protein of embodiment 140, wherein the docking moleculecomprises or is wide type or modified VISTA receptor or PD-1.142. The fusion protein of embodiment 130, wherein the binding domain isa scFv binding PDL1 or PD1, and/or the docking molecule comprises or iswide type or modified PD-1.143. The fusion protein of embodiment 130, wherein the binding domain isa scFv binding B7-H3.144. The fusion protein of embodiment 143, wherein the docking moleculecomprises or is wide type or modified B7-H3 receptor or PD-1.145. A fusion protein comprising a therapeutic agent and a dockingmolecule, wherein the docking molecule comprises a cytoplasmic domainand a transmembrane domain that associate the therapeutic agent with acell.146. The fusion protein of embodiment 142, wherein the therapeutic agentcomprises or is the binding domain of one of embodiment 5-20 or thecytokine of one of embodiments 3 and 4.148. A nucleic acid sequence encoding the fusion protein of one ofembodiments 1-22.147 A modified cell comprises the fusion protein of one of embodiments126-148 or the nucleic acid sequence of embodiment 23.149. A pharmaceutical composition comprising the population of themodified cells of embodiment 24.150. A method of cause T cell response in a subject in need thereofand/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 24 tothe subject.151. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 148-26, wherein the linker is a GS linker.152. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 148-26, wherein the modified cell comprises a CAR.153. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 148-26, wherein the CAR comprises an extracellulardomain, a transmembrane domain, and an intracellular domain, theextracellular domain binds an antigen.154. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 148-26, wherein the intracellular domain comprisesa co-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.155. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 148-26, wherein the antigen is Epidermal growthfactor receptor (EGFR), Variant III of the epidermal growth factorreceptor (EGFRvIII), Human epidermal growth factor receptor 2 (HER2),Mesothelin (MSLN), Prostate-specific membrane antigen (PSMA),Carcinoembryonic antigen (CEA), Disialoganglioside 2 (GD2),Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonic anhydrase IX(CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen 125 (CA125),Cluster of differentiation 133 (CD133), Fibroblast activation protein(FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folatereceptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3,CD5, B-Cell Maturation Antigen (BCMA), or CD4.156. The pharmaceutical composition, the modified cell, and the methodof one of embodiments 126-31, wherein the fusion protein is regulated byan inducible gene expression system.157. A fusion protein comprising a cytokine and an oxygen-sensitivepolypeptide domain.158. The fusion protein of embodiment 157, wherein the oxygen-sensitivepolypeptide domain is s HIFI alpha, HIF3 alpha, or a sequencing havingan identity of over 80%, preferably 90% or more preferably 95% withrespectively Hif VHL-interaction domain or Hif amino acid 344-417 Hifamino acid 380-603.159. The fusion protein of embodiment 158, wherein the oxygen-sensitivepolypeptide domain comprises HIF VHL binding domain.160. The fusion protein of embodiment 157, wherein HIF1 alpha ishydroxylated by HIFa specific prolyl hydroxylases (PHDI-3) which areoxygen sensing. Hydroxylation triggers poly-ubiquitylation of HIF1 alphaand targets the latter for proteosomal degradation by an E3 ubiquitinligase. In hypoxia (low 0 2), occur an inhibition of hydroxylation viaTCA cycle intermediates, a stabilization of the HIFa protein and animpairment of HIF transcriptional activity.161. A nucleic acid sequence encoding the fusion of any of embodiments157-160 or comprising one or more components shown in FIG. 5.162. A modified cell comprising the fusion protein of any of embodiments157-160 and/or the nuclei acid sequence of embodiments 161.163. The cell of embodiment 162, wherein the fusion protein is regulatedby NFAT.164. A pharmaceutical composition comprising a population of themodified cells of any one of embodiments 162 and 163.165. A method of cause T cell response in a subject in need thereofand/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 164to the subject.166. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-165, wherein the modified cell is lymphocyte,leukocyte, or PBMC; or cells, NK cells, or dendritic cells.167. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-166, wherein the modified cell further comprises aChimeric antigen receptor (CAR) or a modified TCR.168. The modified cell, pharmaceutical composition or method ofembodiment 167, wherein the TCR is modified TCR.169. The modified cell, pharmaceutical composition or method ofembodiment 167, wherein the TCR is derived from spontaneously occurringtumor-specific T cells in patients.170. The modified cell, pharmaceutical composition or method ofembodiment 167, wherein the TCR binds a tumor antigen.171. The modified cell, pharmaceutical composition or method ofembodiment 170, wherein the tumor antigen comprises CEA, gp100, MART-1,p53, MAGE-A3, or NY-ESO-1, or the TCR comprises TCRγ and TCRδ Chains orTCRα and TCRβ chains, or a combination thereof.172. The modified cell, pharmaceutical composition or method of claim167, wherein the CAR comprises an extracellular domain, a transmembranedomain, and an intracellular domain, the extracellular domain binds anantigen.173. The modified cell, pharmaceutical composition or method ofembodiment 172, wherein the intracellular domain comprises aco-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and any combination thereof.174. The modified cell, pharmaceutical composition or method ofembodiment 173, wherein the antigen is Epidermal growth factor receptor(EGFR), Variant III of the epidermal growth factor receptor (EGFRvIII),Human epidermal growth factor receptor 2 (HER2), Mesothelin (MSLN),Prostate-specific membrane antigen (PSMA), Carcinoembryonic antigen(CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.175. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-174, wherein the modified cell or the T cellscomprise an additional CAR binding a solid tumor antigen, and the CARbinds an antigen of a white blood cell.176. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-174, wherein the modified cell or the T cellscomprise a dominant negative PD-1.177. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-174, wherein the modified cell or the T cellscomprise a modified PD-1 lacking a functional PD-1 intracellular domain.178. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-177, wherein the modified cell further comprises anucleic acid sequence encoding therapeutic agent.179. The modified cell, pharmaceutical composition or method of any oneof embodiments 178, wherein the isolated nucleic acid sequence comprisesa promoter comprising a binding site for a transcription modulator thatmodulates the expression and/or secretion of the therapeutic agent inthe cell.180. The modified cell, pharmaceutical composition or method ofembodiment 179, wherein the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB.181. The modified cell, pharmaceutical composition or method ofembodiment 180, wherein the promoter is responsive to the transcriptionmodulator.182. The modified cell, pharmaceutical composition or method ofembodiment 180, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives expression and/or secretion of the therapeutic agent in the cell.183. The modified cell, pharmaceutical composition or method ofembodiment 180, wherein expression of the therapeutic agent is regulatedby an inducible gene expression system.184. The modified cell, pharmaceutical composition or method ofembodiment 183, wherein the inducible gene expression system comprisesor is a lac system, a tetracycline system, or a galactose system.185. The modified cell, pharmaceutical composition or method ofembodiment 183, wherein the inducible gene expression system comprisesor is a tetracycline system.186. The modified cell, pharmaceutical composition or method ofembodiment 185, wherein the inducible gene expression system comprisesor is a tetracycline on system, and an inducer is tetracycline,doxycycline, or an analog thereof.187. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-186, wherein the modified cell is a T cell derivedfrom a primary human T cell isolated from a human donor.188. The modified cell, pharmaceutical composition or method ofembodiment 187, wherein the cell has a reduced expression of endogenousTRAC gene.189. The modified cell, pharmaceutical composition or method of any oneof embodiments 162-186, wherein the modified cell is a T cell derivedfrom a primary human T cell isolated from a subject having cancer.190. A composition comprising a first population of cells comprising afirst molecule binding a first antigen and a second population of cellscomprising a second molecule binding a second antigen, wherein thesecond antigen is a tumor antigen and the first antigen and secondantigen are different antigens, and the first population of cells and/orthe second population of cells comprise a nucleic acid sequence encodinga therapeutic agent that is or comprises IL-6 or IFN-γ, or a combinationthereof.191. The composition of embodiment 190, wherein the first molecule is afirst CAR, and the second molecule is a second CAR; or the firstmolecule is the first CAR, and the second molecule is a TCR.192. The composition of embodiment 191, wherein the first population ofcells does not comprise the second CAR, and/or the second population ofcells does not comprise the first CAR.193. The composition of embodiment 192, wherein the composition furthercomprises a third population of cells comprising one or more nucleicacid sequences encoding the first CAR and the second CAR.194. The composition of embodiment 191, wherein:the second population of cells comprises the first CAR, and the firstpopulation of cells do not comprise the second CAR; orthe first population of cells comprises the second CAR.195. The composition of embodiment 194, wherein second population ofcells does not comprise the first CAR, and the first population of cellscomprise the second CAR.196. A method of enhancing expansion of the second population of cells(cells targeting solid tumor), the method comprising administering aneffective amount of the composition of one of embodiments 191-195 to asubject having a form of cancer associated with or expresses the tumorantigen.197. A method of enhancing T cell response in a subject or treating thesubject having cancer, the method comprising administering an effectiveamount of the composition of one of embodiments 191-195 to the subjecthaving cancer associated with or expresses the tumor antigen.198. A method of enhancing expansion of cells in a subject, the methodcomprising:contacting cells with a first vector comprising a first nucleic acidsequence encoding the first CAR and a second vector comprising a secondnucleic acid sequence encoding the second CAR to obtain the compositionof one of embodiments 191-195; andadministering an effective amount of the composition to the subjecthaving a form of cancer associated with or expresses the tumor antigen.199. A method of enhancing T cell response in a subject or treating thesubject having cancer, the method comprising:contacting cells with a first vector comprising a first nucleic acidsequence encoding the first CAR and a second vector comprising a secondnucleic acid sequence encoding the second CAR to obtain the compositionof one of embodiments 191-195; andadministering an effective amount of the composition to the subjecthaving a form of cancer associated with or expresses the tumor antigen.200. A method of enhancing expansion of cells in a subject, the methodcomprising:administering an effective amount of the first population of cells ofone of embodiments 191-195; andadministering an effective amount of the second population of cells.201. The method of one of embodiments 196-200, wherein the first vectorand the second vector comprise lentiviral vectors.202. The composition or the method of one of embodiments 190-201,wherein the first or second antigen is or comprises a surface moleculeof a white blood cell (WBC), a tumor antigen, or a solid tumor antigen.203. The composition or the method of one of embodiments 190-201,wherein the cells are modified T cells, modified NK cells, or modifieddendritic cells.204. The composition or the method of embodiment 202, wherein the WBC isa granulocyte, a monocyte, or lymphocyte.205. The composition or the method of embodiment 204, wherein the WBC isa B cell.206. The composition or the method of embodiment 205, wherein the cellsurface molecule of the WBC is CD19, CD22, CD20, BCMA, CD5, CD7, CD2,CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38,CD138, or CD13.207. The composition or the method of embodiment 202, wherein the cellsurface molecule of the WBC is CD19, CD20, CD22, or BCMA.208. The composition or the method of embodiment 202, wherein the cellsurface molecule of the WBC is CD19.209. The composition or the method of embodiment 202, wherein the tumorantigen is a solid tumor antigen.210. The composition or the method of embodiment 202, wherein the solidtumor antigen is tMUC1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17,TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3, SSTR1,GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119, CLDN6,UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA, EphA2,FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2,EpCAM, EGFRvIII, B7-H3, or EGFR.211. The composition or the method of embodiment 202, wherein the solidtumor antigen is or comprises tumor associated MUC1.212. The composition or the method of one of embodiments 191-211,wherein the CAR comprises the antigen binding domain, a transmembranedomain, a co-stimulatory domain, and a CD3 zeta domain.213. The composition or the method of embodiment 212, wherein theco-stimulatory domain comprises the intracellular domain of CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, or a combination thereof.214. The composition or the method of embodiment 213, wherein:a co-stimulatory domain of the second CAR comprises or is anintracellular domain of 4-1BB, anda binding domain of the second CAR binds TSHR; and/ora binding domain of the first CAR binds CD19 and a co-stimulatory domainof the second CAR comprises or is an intracellular domain of CD28.215. The composition or the method of any one of embodiments 191-214,wherein the first population of cells and/or the second population ofcells further comprise a dominant negative form of PD-1.216. The composition or the method of embodiment 215, wherein the firstpopulation of cells comprise a vector encoding the first CAR and thedominant negative form of PD-1.217. The composition or the method of one of embodiments 191-216,wherein the first CAR comprises a scFv binding TSHR, an intracellulardomain of 4-1BB or CD28, CD3 zeta domain, and the second CAR comprises ascFv binding CD19, an intracellular domain of 4-1BB or CD28, CD3 zetadomain.218. The composition or the method of one of embodiments 191-217,wherein the first CAR comprises SEQ ID NO: 5, and the second CARcomprise the SEQ ID NO: 70.219. The composition or the method of one of embodiments 191-218,wherein the second population of cells comprises a lentiviral vectorencoding the first CAR and a therapeutic agent and the first populationof cells comprises a lentiviral vector encoding the second CAR and adominant negative form of PD-1.220. The composition or the method of one of embodiments 191-219,wherein the first population of cells comprise the first CAR and atherapeutic agent and the second population of cells comprise the secondCAR and a dominant negative form of PD-1.221. The composition or the method of one of embodiments 219 and 220,wherein the therapeutic agent comprises or is a cytokine.222. The composition or the method of embodiment 221, wherein thecytokine is IL6 and/or INFγ.223. A method comprising:administering an effective amount of a first population of T cellscomprising a CAR comprising a scFv binding CD19, an intracellular domainof 4-1BB or CD28, CD3 zeta domain to the patient, thereby enhancingexpansion of the first population of T cells in the patient; andadministering an effective amount of a second population of T cellscomprising a CAR comprising a scFv binding TSHR to a patient havingcancer, an intracellular domain of 4-1BB or CD28, CD3 zeta domain.224. The method of embodiment 223, wherein first population of cellsfurther comprise an additional CAR comprising the scFv binding tMUC1,the intracellular domain of 4-1BB or CD28, and the CD3 zeta domain.225. The method of embodiment 223, wherein the second population ofcells does not comprise the scFv binding CD19.226. The method of embodiment 223, wherein the first population of cellsdoes not comprise the scFv binding TSHR.227. The composition of embodiment 190, wherein the first molecule is amodified TCR.228. The composition of embodiment 227, wherein the TCR is derived fromspontaneously occurring tumor-specific T cells in patients.229. The composition of embodiment 227, wherein the TCR binds a tumorantigen.230. The composition of embodiment 227, wherein the tumor antigencomprises CEA, gp100, MART-1, p53, MAGE-A3, or NY-ESO-1, or the TCRcomprises TCRγ and TCRδ Chains or TCRα and TCRβ chains, or a combinationthereof.231. The modified cell, pharmaceutical composition or method of one ofembodiments 190-230, wherein the modified cell is a T cell derived froma primary human T cell isolated from a human donor.232. The modified cell, pharmaceutical composition or method ofembodiment 231, wherein the cell has a reduced expression of endogenousTRAC gene.233. The modified cell, pharmaceutical composition or method of one ofembodiments 190-230, wherein the modified cell is a T cell derived froma primary human T cell isolated from a subject having cancer.234. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments190-233, wherein the nucleic acid sequence comprises a first nucleicacid sequence encoding IL6 and a second nucleic acid sequence encodingIFN-γ, and the first nucleic acid sequence and the second nucleic acidsequence are connected by an IRES element or a third nucleic acidsequence encoding a 2A peptide.235. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments190-234, wherein the nucleic acid sequence is or comprises the nucleicacid sequence encoding one or more amino acid sequences of SEQ ID NOs:287 and/or 328, or a combination thereof.236. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments190-235, wherein expression of the nucleic acid sequence is regulated bya conditional expression system such that the therapeutic agent isexpressed in response to binding of a target antigen.48 The isolated nucleic acid sequence, the population of CAR cells, thepharmaceutical composition, or the method of one of embodiments 1-47,wherein expression of the additional nucleic acid sequence is regulatedby SynNotch polypeptide.237. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 190-48, wherein T cell response is enhanced as compared tothe administration of T cells that do not express or secrete thetherapeutic agent, or the T cell response is enhanced as compared to theadministration of CAR T cells without the administration of therapeuticagent.238. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 190-49, wherein expression and/or secretion of thetherapeutic agent is regulated by an inducible expression system and/orthe modified cell comprises a nucleic acid sequence encoding aninducible suicide system.239. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiments 190-49, wherein a range of concentration values of IL6 is 60to 5000 pg/ml, 200-5000 pg/ml, or 2000-5000 pg/ml in the blood of thesubject.240. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 190-49, wherein a range of concentration values IFN-γ is 20to 5000 pg/ml, 200 to 5000 pg/ml, or 500 to 5000 pg/ml in the blood ofthe subject.241. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 190-235, wherein the modified cell comprises a nucleic acidsequence comprising a binding site for a transcription modulator thatmodulates the expression and/or secretion of the therapeutic agent inthe cell.242. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiment 241, wherein the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB.55 The isolated nucleic acid sequence, the population of CAR cells, thepharmaceutical composition, modified cell, or the method of embodiment53, wherein the promoter is responsive to the transcription modulator.243. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiment 241, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives 57 and/or secretion of the therapeutic agent in the cell.244. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 241-56, wherein the promoter comprises at least one of SEQID Nos: 332, 333, 341, 469, or 342.245. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 190-57, wherein the first and second population of cellscomprises TSHR-CAR (scFv of the CAR: SEQ ID NO: 8): andhCD19-CAR-NATF-IL6-2A-IFNγ (scFv of CD19 CAR: SEQ ID 5, aa of NATF: SEQID: 469, aa of IL6: SEQ ID NO: 287, 2A is SEQ ID NO: 326, and aa ofIFN-γ: SEQ ID NO: 327). SEQ ID NO: 287, 2A is SEQ ID NO: 327, and aa ofIFN-γ: SEQ ID NO: 328).246. An isolated nucleic acid sequence comprising a nucleic acidsequence and an additional nucleic acid sequence, the nucleic acidsequence encoding a chimeric antigen receptor (CAR), the additionalnucleic acid sequence encoding a therapeutic agent that is or comprisesIL-6 or IFN-γ, or a combination thereof.247. A population of CAR cells comprising the nucleic acid sequence andthe additional nucleic acid sequence of embodiments 246, wherein the CARcells comprise lymphocyte, leukocyte, or PBMC.248. The population of CAR cells of embodiment 247, wherein the CAR andthe therapeutic agent are produced in the form of a polyprotein, whichis cleaved to generate separate CAR and therapeutic agent molecules.249. The population of CAR cells of one of embodiments 247-248, whereinthe polyprotein comprises a cleavable moiety between the CAR and thetherapeutic agent, the cleavable moiety comprises a 2A peptide, the 2Apeptide comprises P2A or T2A, and/or the CAR and the therapeutic agentare each constitutively expressed.250. The population of CAR cells of one of embodiments 247-249, whereinthe CAR cells comprise:a third nucleic acid sequence encoding an additional CAR binding to anantigen that is different from an antigen that the CAR binds, orthe additional CAR binding a solid tumor antigen, and the CAR binds anantigen of a white blood cell.251. A pharmaceutical composition comprising the population of the CARcells of one of embodiments 247-250.252. A method of causing T cell response in a subject in need thereofand/or treating a tumor of the subject, the method comprisingadministering an effective amount of the composition of embodiment 251to the subject.253. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments246-252, wherein the additional nucleic acid sequence comprises a firstnucleic acid sequence encoding IL6 and a second nucleic acid sequenceencoding IFN-γ, and the first nucleic acid sequence and the secondnucleic acid sequence are connected by an IRES element or a thirdnucleic acid sequence encoding a 2A peptide.254. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments246-253, wherein the additional nucleic acid sequence is or comprisesthe nucleic acid sequence of SEQ ID NOs: 287 or 328, or a combinationthereof.255. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, or the method of one of embodiments246-254, wherein expression of the additional nucleic acid sequence isregulated by a conditional expression system such that the therapeuticagent is expressed in response to binding of a target antigen.11 The isolated nucleic acid sequence, the population of CAR cells, thepharmaceutical composition, or the method of one of embodiments 1-10,wherein expression of the additional nucleic acid sequence is regulatedby SynNotch polypeptide.256. A modified cell comprises one or more CARs, wherein the cell isengineered to express and secrete a therapeutic agent that is orcomprises IL-6 or IFN-γ, or a combination thereof.257. A method of causing or enhancing T cell response, treating cancer,or enhancing cancer treatment, the method comprising: administrating aneffective amount of the composition of T cells comprising one or moreCARs, wherein the cell is engineered to express and secrete atherapeutic agent that is or comprises IL-6 or IFN-γ, or a combinationthereof.258. A method of causing or enhancing T cell response, treating cancer,or enhancing cancer treatment, the method comprising:administering an effective amount of the composition of a population ofT cells comprising a CAR; andadministering an effective amount of a therapeutic agent that is orcomprises IL-6 or IFN-γ, or a combination thereof.259. The modified cell or the method of one of embodiments 252, 257, and258, wherein T cell response is enhanced as compared to theadministration of T cells that do not express or secrete the therapeuticagent, or the T cell response is enhanced as compared to theadministration of CAR T cells without the administration of therapeuticagent.260. The modified cell or the method of one of embodiments 256-259,wherein expression and/or secretion of the therapeutic agent isregulated by an inducible expression system and/or the modified cellcomprises a nucleic acid sequence encoding an inducible suicide system.261. The modified cell or the method of one of embodiments 256-259,wherein a range of concentration values of IL6 is 60 to 5000 pg/ml,200-5000 pg/ml, or 2000-5000 pg/ml in the blood of the subject.262. The modified cell or the method of one of embodiments 256-259,wherein a range of concentration values IFN-γ is 20 to 5000 pg/ml, 200to 5000 pg/ml, or 500 to 5000 pg/ml in the blood of the subject.263. The modified cell or the method of one of embodiments 256-262,wherein the administering the effective amount of the therapeutic agentcomprises intravenous delivery of an amount of human IL-6 in the rangeof about 0.5-50 ug per kilogram of body weight.264. The modified cell or the method of one of embodiments 256-263,wherein the modified cell or the T cells comprise an additional CARbinding a solid tumor antigen, and the CAR binds an antigen of a whiteblood cell.265. The modified cell or the method of embodiment 264, wherein thesolid tumor antigen is tMUC 1, PRLR, CLCA1, MUC12, GUCY2C, GPR35, CR1L,MUC 17, TMPRSS11B, MUC21, TMPRSS11E, CD207, SLC30A8, CFC1, SLC12A3,SSTR1, GPR27, FZD10, TSHR, SIGLEC15, SLC6A3, KISS1R, QRFPR, GPR119,CLDN6, UPK2, ADAM12, SLC45A3, ACPP, MUC21, MUC16, MS4A12, ALPP, CEA,EphA2, FAP, GPC3, IL13-Ra2, Mesothelin, PSMA, ROR1, VEGFR-II, GD2, FR-α,ErbB2, EpCAM, EGFRvIII, PSCA, or EGFR, and the B cell antigen is CD19,CD20, CD22, or BCMA.266. The modified cell or the method of one of embodiments 256-265,wherein the modified cell or the T cells comprise a dominant negativePD-1.22. The modified cell or the method of one of embodiments 12-21, whereinthe modified cell or the T cells comprise a modified PD-1 lacking afunctional PD-1 intracellular domain.268. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-23, wherein the CAR comprises an extracellular domain, atransmembrane domain, and an intracellular domain, the extracellulardomain binds an antigen.269. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-23, wherein the intracellular domain comprises aco-stimulatory domain that comprises an intracellular domain of aco-stimulatory molecule selected from the group consisting of CD27,CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3,and one combination thereof.270. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-24, wherein the antigen is Epidermal growth factorreceptor (EGFR), Variant III of the epidermal growth factor receptor(EGFRvIII), Human epidermal growth factor receptor 2 (HER2), Mesothelin(MSLN), Prostate-specific membrane antigen (PSMA), Carcinoembryonicantigen (CEA), Disialoganglioside 2 (GD2), Interleukin-13Ra2 (IL13Rα2),Glypican-3 (GPC3), Carbonic anhydrase IX (CAIX), L1 cell adhesionmolecule (L1-CAM), Cancer antigen 125 (CA125), Cluster ofdifferentiation 133 (CD133), Fibroblast activation protein (FAP),Cancer/testis antigen 1B (CTAG1B), Mucin 1 (MUC1), Folate receptor-α(FR-α), CD19, FZD10, TSHR, PRLR, Muc 17, GUCY2C, CD207, CD3, CD5, B-CellMaturation Antigen (BCMA), or CD4.271. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-25, wherein the therapeutic agent is present in themodified cell in a recombinant DNA construct, in an mRNA, or in a viralvector.272. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-26, wherein the modified cell comprises a therapeuticagent mRNA encoding the therapeutic agent, and the mRNA is notintegrated into the genome of the modified cell.273. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 246-27, wherein the modified cell comprises a nucleic acidsequence comprising a promoter comprising a binding site for atranscription modulator that modulates the expression and/or secretionof the therapeutic agent in the cell.274. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiment 273, wherein the transcription modulator is or includesHif1a, NFAT, FOXP3, and/or NFkB.275. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiment 273, wherein the promoter is responsive to the transcriptionmodulator.276. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method ofembodiment 273, wherein the promoter is operably linked to the nucleicacid sequence encoding the therapeutic agent such that the promoterdrives expression and/or secretion of the therapeutic agent in the cell.277. The isolated nucleic acid sequence, the population of CAR cells,the pharmaceutical composition, modified cell, or the method of one ofembodiments 273-276, wherein the promoter comprises at least one of SEQID Nos: 323-325.278. A method of using any one of the preceding embodiments (1-277) inautologous T cell therapy, allogenic T cell therapy, TCR T cell therapy,or NK cell therapy.279. The CAR described in any one of the preceding embodiments (1-278),wherein the CAR comprises one or more of the complementarity determiningregions (CDRs) that binds an antigen of interest.

Examples In Vivo Expansion and Treatment of Cancer

These clinical studies were designed to assess the safety and efficacyof infusing autologous T cells modified to express TSHR specificCAR/4-1BB/CD3-ζ into patients. On the first arm of the studies, patientsreceived TSHR CART cells only. On the second arm, patients received CARTcells directed to CD19 and TSHR. Autologous T cells modified to expressTSHR specific CAR/4-1BB/CD3-ζ (TSHR CAR) and CD19 specificCAR/4-1BB/CD3-ζ (CD19 CAR) were infused into a patient. The modified Tcell included T cells expressing TSHR CAR (single CAR), CD19 CAR (singleCAR), and TSHR CAR&CD19 CAR, respectively (Double CAR). T cells of thepatients were obtained, modified, and infused to the patients. T cellresponses of patients from the first and second arms were measured andcompared using the following protocols, which were approved by thehospitals where the trials were conducted. All patients were providedwith written informed consent (SD: stable disease; PD: progressivedisease; PR: partial remission; CR: complete remission; NR, noresponse).

TABLE 5 Safety or Patient Target Infusion Vectors mixed with AdverseCancer ID Marker CART/kg T cells reaction (AR) Efficacy Thyroid 001 TSHR1.1 × 10⁶ Vectors encoding No apparent AR PR (day cancer TSHR-CAR & 29)vectors encoding CR (day CD19-CAR 64) Thyroid 002 TSHR 1.1 × 10⁶ Vectorencoding No apparent AR NR cancer TSHR-CAR

Manufacturing of CAR T Cells

PBMCs were obtained from patients. Various lentiviral vectors weregenerated and then transfected to the T cells, which were furthercultured for several days before the co-cultivation assay. Moreinformation may be found in Table 6 below. Techniques related to cellcultures and cytotoxic T-lymphocyte assay may be found in “Control oflarge, established tumor xenografts with genetically retargeted human Tcells containing CD28 and CD137 domains,” PNAS, Mar. 3, 2009, vol. 106no. 9, 3360-3365, which is incorporated herein by reference in itsentirety.

TABLE 6 P's Infusion ID Vectors and MOI Methods Pre-treatment 001TSHR-CAR (CAR: SEQ ID NO: 279; Fresh FC regimen at −5 to −3 days scFv ofthe CAR: SEQ ID NO: 8): 19:1 cells (cyclophosphamide 500 mg/m2,hCD19-CAR-NATF-IL6-2A-IFNγ (scFv fludarabine 30 mg/m2) of CD19 CAR: SEQID 5, 6xNFAT: SEQ ID: 469, aa of IL6: SEQ ID NO: 287, 2A is SEQ ID NO:326, and aa of IFN-γ: SEQ ID NO: 327 (See Embodiment 1 of FIG. 10)): 5:1002 TSHR-CAR (CAR: SEQ ID NO: 279; Fresh FC regimen at −5 to −3 daysscFv of the CAR: SEQ ID NO: 8): 19:1 cells (cyclophosphamide 500 mg/m2,fludarabine 30 mg/m2)

PBMCs were cultured using TEXMACS culture containing IL-2. CD4, and CD8magnetic beads were used to sort and select T cells in the PBMCs. Theappropriate starting culture amount was selected and TransAct activatorwas used to activate T cells. MACS® GMP T Cell TransAct™ includes acolloidal polymeric nanomatrix covalently attached to humanizedrecombinant agonists against human CD3 and CD28. Due to the nanomatrixMACS GMP T Cell TransAct can be sterile filtered and excess reagent canbe removed by centrifugation and following conventional supernatantreplacement or simply by medium wash. This reagent is suitable for usein automated culture systems, such as the CliniMACS Prodigy® Instrument.The number of corresponding carriers and the volume of the carrier werecalculated according to the required carrier MOI (See Table 6). Variousassays were performed to confirm the efficacy of these CART cells (e.g.,a binding assay using flow cytometry and a killing assay using culturingassay with cells expressing TSHR/CD19), and quality assurance procedureswere followed to ensure the safety of the administration of the CAR Tcells to the patient.

FIG. 18 shows copy numbers of T cells expressing various proteins. Day1, T cells from healthy donor were sorted and activated using CD3/CD28beads. Day 2, 10⁵ cells were transfected with vectors 1230 (MOI 10:1),6205 (hCD19CAR-GFP) (MOI 60:1), and 6221 (hCD19CAR-6×NFAT-IL6-2a-IFNg)(MOI 60:1), respectively. On Day 3, cell media were changed. On Day 4,cell numbers were counted. On Days 5, 6, and 8, assays for measuringculturing factors, CAR copy number, phenotype and expression wereconducted. Day 8, toxicity assays were performed, and culturing factorswere detected. The copy numbers are provided in Table 7 below. In thisand the following examples, sequences for NFAT is SEQ ID NO 469; forTSHR-CAR is SEQ ID NO: 279; for scFv of TSHR CAR is SEQ ID NO: 8; forscFv of CD19 CAR is SEQ ID 5; for IL6 is SEQ ID NO: 287; for 2A is SEQID NO: 327; and for IFN-γ is SEQ ID NO: 328. Sequences of othercomponents may be found in Table 2.

Table 7 shows copy numbers of CAR per CAR T cell.

Type of T cells 6205 6221 1230 Day8/per CAR-T 1.02728 0.66634 1.3325

FIG. 19 shows flow cytometry assay results of T cells expressing variousproteins shown in FIG. 18. Day 0, peripheral blood of healthy volunteerswas taken; CD3+ T cells were sorted; and CD3/CD28 Dynabeads were addedin a 1:1 ratio. Day 2, T cells were transfected using lentivirusincluding various following vectors. CD19CAR was infected according tothe infection ratio of M01=10:1; hCD19CAR, hCD19CAR-6×NFAT-GFP,hCD19CAR-6×NFAT-GFP, hCD19CAR-6×NFAT-IL6-2a-IFNg cells were infectedaccording to the infection ratio of M01=60:1. Day 3, the media werechanged, the lentivirus were removed, and the cells were resuspended infresh medium. Day 7, flow cytometry assays were used to detect CARexpression. CD19CAR is a humanized antibody and is therefore detectedwith a human CAR antibody. As shown in FIG. 19, CD19CAR expression was23.99%; hCD19CAR-6×NFAT-GFP CAR expression was 25%; andhCD19CAR-6×NFAT-IL6-2a-IFNg CAR expression was 17.6%. Flow cytometryassay was performed using human CAR antibody to detect the expressionintensity and expression level of CAR.

FIG. 20 shows IL6 release in response to CD3/CD28 Dynabeads activation.Day 0, peripheral blood of healthy volunteers was taken, CD3+ T cellswere sorted, and CD3/CD28 Dynabeads were added at a 1:1 ratio. Day 2, Tcells were transfected using lentivirus including various followingvectors. CD19CAR was infected according to the infection ratio ofM01=10:1; hCD19CAR, hCD19CAR-6×NFAT-GFP, hCD19CAR-6×NFAT-GFP,hCD19CAR-6×NFAT-IL6-2a-IFNg cells were infected according to theinfection ratio of M01=60:1. Day 3, the media were changed, and thelentiviruses were removed, and the cells were resuspended in freshmedium. DAY 5, 6, and 8, the cell supernatant in 200 ul culture was usedto detect the release of IL6. As shown in FIG. 20, on Day 5, 6, and 8,the cell supernatant of 200 ul was taken from the media and the releaseof IL6 factor was detected. The amount of IL6 released per 10⁴ ofCD19CAR and CD19CAR-6×NFAT-GFP was 0-10 pg/ml on Day 5, 6, and 8. 10⁴CD19CAR-6×NFAT-1L6-2A-IFNg had IL6 release of 498 pg/ml and 378 pg/ml onDay 5 and 6, and the released amount of IL6 on Day 8 was 9.8 pg/ml. OnDays 5 and 6, cells were cultured with CD3/CD28 Dynabeads such that thecells were activated, and NFAT element was activated and thetranscription of IL6 was enabled, causing IL6 to be released. However,on Day 8, the effect of Dynabeads stimulation was dropped to a lowerlevel, and the cells were not activated. Therefore, the NFAT element wasturned off and the transcription of IL6 was disabled. Thus, IL6 was notreleased. When T cells are stimulated by CD3/28 Dynabeads, the cells areactivated for a short period of time. The NFAT element inducestranscriptional translation of the gene in response to the activation,and the corresponding genes are expressed. When the cells are at rest orat a lower level of activation, the NFAT element does not initiatetranscriptional translation of the gene of interest. Therefore, whetherthe NFAT element is active or not and whether the induced gene isexpressed can be judged by the expression of the target gene in theactivated and inactivated state of the CART cell.

FIG. 21 shows IL6 release in response to co-culturing with Nalm6 cells(See Table below). The cells were cultured to Day 8 and then wereleveled with NT cells to differentiate the CAR ratio of CD19CAR T cellsand CD19CAR-6×NFAT-GFP to cells of CD19CAR-6×NFAT-IL6-2a-IFNg. 10⁴ CAR+cells were co-cultured with 10⁴ Nalm-6 cells or cultured separately.After 24H, the supernatant was collected and the amount of IL6 releasedwas measured. The cells were co-cultured with Nalm6 cells and were in anactivated state. Thus, the NFAT element initiated transcription of IL6in an activated state, allowing IL6 to be released. When the CAR T cellsare co-cultured with the target cells, since the CAR T cells recognizethe membrane proteins on the surface of the tumor cells, the cells areactivated. The NFAT element initiates transcriptional translation of thegene in response to activation. The corresponding gene is expressed.When the cell is at rest or the activation level is low, the NFATelement does not initiate transcriptional translation of the gene ofinterest. Therefore, whether the NFAT element is active or not andwhether the induced gene is expressed can be determined by theexpression of the target gene in the activated and inactivated state ofthe CART cell.

TABLE 8 CD19CAR-6xNFAT- CD19CAR-6xNFAT- CAR T cells 10⁴ CD19CAR-T GFPIL6-2a-IFNg Co-cultured cells (25 Nalm6 Nalm6 Nalm6 hours) 10⁴ IL6released: CAR T 0-10 pg/ml 0-10 pg/ml 260 pg/ml cells co-cultured withNalm6 IL6 released: CAR T 0-10 pg/ml 0-10 pg/ml 0-10 pg/ml cellscultured alone

FIG. 22 shows IFNγ (i.e., IFNg) release in response to CD3/CD28Dynabeads activation. On day 0, peripheral blood of healthy volunteerswas taken, CD3+ T cells were sorted, and CD3/CD28 Dynabeads were addedin a 1:1 ratio. On Day 2, T cells were transfected using lentivirusincluding various following vectors. CD19CAR T cells were infectedaccording to the infection ratio of M01=10:1; hCD19CAR,hCD19CAR-6×NFAT-GFP, hCD19CAR-6×NFAT-GFP, hCD19CAR-6×NFAT-IL6-2a-IFNgcells were infected according to the infection ratio of M01=60:1. Thelentivirus was removed, and the cells were resuspended in fresh medium.On DAY 5, 6, and 8, 200 ul of the cell supernatant was used to detectthe release of IFNγ. On Days 5 and 6, cells were cultured with CD3/CD28Dynabeads such that the cells were activated, and NFAT element wasactivated which enabled the transcription of IFNγ and the release ofIFNγ. However, on Day 8, the effect of Dynabeads stimulation has droppedto a lower level, and the cells were no longer activated. Therefore, theNFAT element was turned off and transcription of IFNγ was disabled.Thus, IFNγ was not released. When T cells are stimulated by CD3/28Dynabeads, the cells are activated for a short period of time. The NFATelement causes transcriptional translation of the gene in response tothe activation, and the corresponding genes are expressed. When thecells are at rest or at a lower level of activation, the NFAT elementdoes not initiate transcriptional translation of the gene of interest.Therefore, whether the NFAT element is active or not and whether theinduced gene is expressed can be determined by the expression of thetarget gene in the activated and inactivated state of the CAR-T cell.

FIG. 23 shows IFNγ release in response to co-culturing with Nalm6 cells(See Table below). The cells were cultured to Day 8 and then wereleveled with NT cells to differentiate the CAR ratio of CD19 CAR T cellsand CD19CAR-6×NFAT-GFP to cells of CD19CAR-6×NFAT-IL6-2a-IFNg. 10⁴ CAR+cells were co-cultured with 10⁴ Nalm-6 cells or cultured separately.After 24 hrs, the supernatant was collected, and the amount of IFNγreleased was measured. The cells were co-cultured with Nalm6 cells andwere in an activated state. Thus, the NFAT element initiatedtranscription of IFNγ in an activated state, allowing IFNγ to bereleased. When the CAR T cells are co-cultured with the target cells,the cells are activated because the CAR T cells recognize the membraneproteins on the surface of the tumor cells. The NFAT element initiatestranscriptional translation of the gene to be expressed due to theactivation. The corresponding gene is expressed. When the cell is atrest or the activation level is low, the NFAT element does not initiatetranscriptional translation of the gene of interest. Therefore, whetherthe NFAT element is active or not and whether the induced gene isexpressed can be determined by the expression of the target gene in theactivated and inactivated state of the CART cell.

TABLE 9 CD19CAR-6xNFAT- CD19CAR-6xNFAT- CAR T cells 10⁴ CD19CAR-T GFPIL6-2a- IFNγ Co-cultured cells (25 Nalm6 Nalm6 Nalm6 hours) 10⁴ IFNγreleased: CAR T 2400-3200 pg/ml 2400-3200 pg/ml 7900 pg/ml cellsco-cultured with Nalm6 IFNγ released: CAR T 2400-3200 pg/ml 2400-3200pg/ml 2400-3200 pg/ml cells cultured alone

FIG. 24 shows toxicity assay with respect to CAR T cells. T cells from ahealthy donor were cultured to Day 8 and then were leveled with NT cellsto differentiate the CAR ratio of CD19 CAR T cells andCD19CAR-6×NFAT-GFP to cells of CD19CAR-6×NFAT-IL6-2a-IFNg. 30e4 CAR+cells were co-cultured with 10⁴ Nalm-6 cells and 90e4 Nalm-6 cells,respectively. The residual of Nalm6 cells was detected after 24 hrs.CD19CAR T cells, CD19CAR-6×NFAT-GFP cells, andCD19CAR-6×NFAT-IL6-2a-IFNg cells were co-cultured with Nalm6 cells indifferent ratios. There were no significant differences among the 3 cellgroups. After the cells of CD19CAR, CD19CAR-6×NFAT-GFP andCD19CAR-6×NFAT-IL6-2a-IFNg were activated by the tumor, the T cellsexecuted a killing function and acted on the target cells to cause thetarget cells to be killed.

FIGS. 25 and 26 show other IFNγ release in response to co-culturing withNalm6 cells. Day 0, peripheral blood T cells were obtained fromvolunteer and stimulated using Dynabeads at 1:1 ratio. Day 1 the cellswere infected with lentiviral vectors. Day 2, the media were changed.Day 7 flow cytometry assays were used to detected CAR expression and CARcopy numbers. 1204 represents hCD19CAR cells, and 6107 representshCD19CAR-2A-IL12 cells which express IL12 continuously. The CARexpression was normalized to 17%. 10⁴ CAR positive cells and 10⁴ Nalm6or Nalm6-PDL1 tumor cells were co-cultured in 24-well plates for 24 h,and the supernatant was assayed for IFNγ. 1204 had 42% CAR expressionand 1.5 copies per CART cell, and 6107 had 23% CAR expression and 0.94copies per CAR T cell. CAR was normalized to 17% for co-culture.Co-culture results, as shown in the histogram, showed that IFNγ producedby Nalm6-PDL1 stimulation for 1204 was about half that stimulated byNalm6 wt because of the inhibitory effect of PDL1 on T cells. Therelease of IFNγ from 6107 reached about 10 times that of 1230,demonstrating that IL12 released by CART significantly promoted IFNγrelease.

FIGS. 27 and 28 show IL12 and IFNγ release in response to CD3/CD28Dynabeads activation. Day 0, peripheral blood T cells were obtained fromvolunteer and stimulated using Dynabeads at 1:1 ratio. Day 1, the cellswere infected with lentiviral vectors. Day 2, the media were changed. Onday 9, flow cytometry was used to detect CAR expression. 1230 representsh19CAR, and 6209 represents h19CAR-6×NFAT-IL12 (conditional release ofIL12 under T cell activation). The CAR expression was normalized to 30%.The same number of cells were stimulated by Dynabeads for 24 hrs at aratio of 1:3, and the supernatant was assayed for IL12 and IFNγ. Therewas 65% CAR expression in 1230 and 34% CAR expression in 6209. Afternormalized to 30%, add Dynabeads. After 24 hrs, the supernatant wasassayed to collect information of cytokines and results were presentedas a histogram. Under the stimulation of Dynabeads, 6209 released anaverage of 55 pg of IL12 per 10x⁴ of T cells. 1230 did not release IL12regardless of stimulation, and 6209 did not release IL12 withoutstimulation. This indicates that, originally, NFAT activated IL12transcription under Dynabeads stimulation, as shown on the left. IFN wasreleased as shown on the right. Both CAR T cells were free of IFNγrelease without stimulation, and 6209 released more IFNγ under Dynabeadsstimulation, indicating that IL12 synergizes with 6209CART cells torelease more IFNγ.

FIGS. 29 and 30 show IL6 and IFNγ release in response to CD3/CD28Dynabeads activation. Day 0, peripheral blood T cells were obtained fromvolunteer and stimulated using Dynabeads at 1:1 ratio. Day 1, the cellswere infected with lentiviral vectors. Day 2, the media were changed. Onday 7, flow cytometry was used to detect CAR expression. 1230 representshCD19CAR, and 6221 represents hCD19CAR-6×NFAT-IL6-2A-IFNγ (conditionalrelease of IL6 and IFNγ under T cell activation). The CAR expression wasnormalized to 12.66%. The same number of cells were stimulated byDynabeads for 24 hrs at a ratio of 1:3, and the supernatant was assayedfor IL6 and IFNγ. There was 67% CAR expression in 1230, and 29% CARexpression in 6221. The CAR expression was normalized 12.66% and thecells were added Dynabeads. After 24 hrs, the supernatant was assayed tocollect information of cytokines and results were presented as ahistogram. Both cells were free of IL6 or IFNγ release without Dynabeadactivation. After addition of Dynabeads, 6221 significantly releasedmore IL6 and IFNγ (based on the average number of pg released per 10⁴ Tcells).

Cell Expansion and Treatment of Cancer

For fresh cells, after removing the magnetic beads, the transduced cellswere centrifuged or replaced with a solution of 95% compound electrolyteinjection of 5% human albumin, loaded into a return bag, and transportedat 15-25° C. after sealing. Fresh preparations were returned directly.For Cryopreserved cells, the transduced cells were transferred to themedia including a compound electrolyte injection of 33.75% human albumin25% dextran 40 glucose injection 33.75% DMSO 7.5%. The cell suspensionwas loaded into a cryopreservation bag and then the procedure was cooledto −90° C. and transferred to a gas phase liquid nitrogen tank forstorage. The reconstitution of the frozen preparations was completedwithin 30 minutes after resuscitation. Peripheral blood mononuclearcells (PBMCs) were obtained from patients by leukapheresis for CAR Tcell preparation, and the first day of CAR T infusion was set as studyday 0.

Patients were given a conditioning treatment for lymphodepletion.Fludarabine- and cyclophosphamide-based conditioning treatment variedaccording to the tumor burden in the bone marrow (BM) and peripheralblood (PB). CAR T cells were transfused to patients. Each day CAR Tcells were transported to the hospital, washed, counted, checked forviability and then prepared for administration to patients, who werethen observed closely for at least 2 hours. Cytokine Release Syndrome(CRS) was graded according to a revised grading system (See Lee D W. etal, Blood 2014; 124:188-95). Other toxicities during and after therapywere assessed according to the National Institutes of Health CommonTerminology Criteria for Adverse Events Version 4.0(http://ctep.cancer.gov/). Therapy responses were assessed by flowcytometry and morphological analysis. When possible, patients wereassessed by chimeric gene expression levels. The response type wasdefined as minimal residual disease (MRD) negative, complete response,complete response with incomplete count recovery, stable disease, andprogressive disease.

Serial BM and PB samples after CAR T cell infusion were collected inK2EDTA BD vacutainer tubes (BD). The persistence of CD19CAR T cells fromfresh PB and BM in patients was determined by FACS. Circulating CAR Tcell numbers per μl were calculated on the basis of measured absoluteCD3+ T lymphocyte counts. Simultaneously, CAR DNA copies were evaluatedas another method of determining CAR T cell expansion and persistence.Genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen)from cryopreserved PB and BM. CAR DNA copies were assessed byquantitative real-time PCR as described in the supplementary materials.The levels of cytokines IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17, etc. inserum and CSF were measured in a multiplex format according to themanufacturer's instructions (See FIGS. 12-15 and 16 as well as 17). OnDay 64, PET CT scanning was performed to evaluate CAR T therapy onpatient 001. Patient 001 had undergone thyroidectomy. The CT imagesshowed that there was no clear tumor recurrence or recurrence in thesurgical area. Thyroid cancer changed the bilateral thyroid surgery areaafter surgery, and no abnormal CT signal was observed in the area. Afterthe scanning signal is enhanced, no abnormal enhancement signal isobserved in the above areas. The double neck II and III areas showedmultiple small lymph nodes with a maximum short diameter of no more than10 mm. There were no abnormalities in the bilateral submandibular glandmorphology and CT signal. At the same time, the cervical spinal cordmorphology and CT signal were not abnormal. It appeared that patient hasachieved complete remission (CR). FIG. 16 shows various parameters ofthe patient in response to CART cell infusion in a patient. FIG. 17shows various parameters of the patient in response to CART cellinfusion in another patient. These results demonstrate that T cellsexpressing CD19 CAR and TSHR CAR expanded more and released morecytokines (e.g., IL-6 an IFN-γ) than T cells expressing only the TSHRCAR.

As for Patient 002, after cell infusion, while the patient did not haveany adverse reaction, no apparent response (e.g., disappearance orshrink of target lesions) was observed. As for Patient 001, 29 daysafter the infusion, the right tumor disappeared, and the size of theleft tumor reduced (See FIG. 11). These results demonstrate that T cellsexpressing CD19 CAR, TSHR CAR, and IL6/IFNγ enhanced or inhibited thegrowth of thyroid cancer. During the treatment, no severe CRS (nogreater than level 2) was observed in Patient 001. This demonstratedthat infusion of CAR T cells expressing and secreting IL-6 did not causesevere CRS for treating solid tumors.

Genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen)from cryopreserved peripheral blood and bone marrow. Quantitative PCR(qPCR) was performed in real-time in triplicates using the ABI 2×TaqManUniversal Master Mix with AmpErase UNG (Applied Biosystems) in a 7500real-time PCR system (Applied Biosystems). Copy numbers per microgram ofgenomic DNA were calculated from a standard curve of 10-fold serialdilutions of purified CAR plasmid containing 102-108 copies/μL.Amplification of an internal control gene was used for normalization ofDNA quantities (See FIG. 16). Primers/probes specific for the CAR19transgene and an internal control gene were as previously described (seeGokbuget N. et al., Blood 2012; 120:2032-41 and O'Brien S. et al, J ClinOncol 2013; 31:676-83).

Cells Expressing Chimeric Receptors Establish Antitumor Effects inPatients with Relapsed/Refractory Acute Lymphocytic Leukemia

This clinical trial was designed to assess the safety and efficacy ofinfusing autologous T cells modified to express CD19 specificCAR/4-1BB/CD3-ζ into Chinese patients with R/R ALL. The inclusioncriteria were as follows: 1) age not more than 60 years; 2) relapsed orrefractory CD19+ ALL; 3) relapsed allo-HSCT without evidence of graftversus host disease (GVHD) and not requiring immunosuppression therapy;and 4) measurable disease and adequate performance status and organfunction. Patients with central nervous system leukemia (CNSL) wereineligible. The protocol was approved by the Institutional Review Board.All patients provided written, informed consent.

The single chain fragment variable (scFv) sequence specific for CD19 wasderived from Clone FMC63 (See Zola H. et al, Immunol Cell Biol 1991;69:411-22.). The 4-1BB co-stimulatory domain, CD3ζ signaling domain andhinge and transmembrane domain were generated. CART19-4-1BB vectorsharboring anti-CD19 scFv (SEQ ID: 6) and the human 4-1BB and CD3ζsignaling domains were cloned into a lentiviral backbone as previouslydescribed (See Hu Y. Journal of Hematology & Oncology 2016; 9: 70).

Lentivirus was produced by transfecting 293T cells with CART19-4-1BBvectors and viral packaging plasmids which were frozen in −80° C. andthawed immediately before transduction. The lentivirus supernatant washarvested. CD3+ T cells were isolated and activated as described (SeeKalos M. et al, Sci Transl Med 2011; 3:95ra73). The cells were thencultured in X-VIVO 15 medium (Lonza) containing 100 U/ml interleukin-2(IL-2) and transduced with lentivirus supernatant at high multiplicityof infection (MOI) from 5:1 to 10:1 within 24-48 hours. The CARtransduced T cells (CD19-CAR T cell, thereafter “CART19”) were obtainedand cultured for 11 days. Three days before administration, freshculture media were replaced. After that, no manipulation was conductedto the cells until transportation for infusion. The transductionefficiency was evaluated by flow cytometry (FACS) on day 5-7 afterlentivirus transduction. The following anti-human antibodies were used:anti-hCD45 APC (BD Bioscience), anti-hCD3 FITC (BD Bioscience),biotin-labeled goat-anti-mouse IgG specific for F(ab′)2 fragment(Jackson immuno-Research, Cat #115-065-072) and PE streptavidin (BDBioscience). Data acquisition was performed using a CytoFLEX flowcytometer (Beckman).

Prior to CD19CAR T infusion, FACS analysis of transduction efficiencyand in vitro cytotoxicity assays of CD19 CAR T were performed for eachpatient as described herein. Additionally, CD19CAR T cultures werechecked twice for possible contaminations by fungus, bacteria,mycoplasma, chlamydia and endotoxin. Peripheral blood mononuclear cells(PBMCs) were obtained from patients by leukapheresis for CD19CAR Tpreparation on day 8, and the first day of CD19CAR T cell infusion wasset as study day 0. Patients were given a conditioning treatment forlymphodepletion. Fludarabine- and cyclophosphamide-based conditioningtreatment varied according to the tumor burden in the bone marrow (BM)and peripheral blood (PB). CD19CAR T cells were transfused directly topatients in escalating doses over a period of 3 consecutive days withoutany premedication. Each day CD19CAR T cells were transported tohospital, washed, counted, checked for viability and then prepared foradministration to patients, who were then observed closely for at least2 hours. CRS was graded according to a revised grading system (See Lee DW. et al, Blood 2014; 124:188-95). Other toxicities during and aftertherapy were assessed according to the National Institutes of HealthCommon Terminology Criteria for Adverse Events Version 4.0(http://ctep.cancer.gov/). Therapy responses were assessed by flowcytometry and morphological analysis. When possible, patients wereassessed by chimeric gene expression levels. The response type wasdefined as minimal residual disease (MRD) negative, complete response,complete response with incomplete count recovery, stable disease andprogressive disease.

Serial BM and PB samples after CD19CAR T cell infusion were collected inK2EDTA BD vacutainer tubes (BD). The persistence of CD19CAR T cells fromfresh PB and BM in patients was determined by FACS. Circulating CD19CART cell numbers per μl were calculated on the basis of measured absoluteCD3+ T lymphocyte counts. Simultaneously, CAR DNA copies were evaluatedas another method of determining CD19CAR T cell expansion andpersistence. Genomic DNA was extracted using a QIAamp DNA Blood Mini Kit(Qiagen) from cryopreserved PB and BM. CAR DNA copies were assessed byquantitative real-time PCR as described in the supplementary materials.

The levels of cytokines such as IFN-γ, TNF-α, IL-4, IL-6, IL-10, IL-17,etc. in serum and CSF were measured in a multiplex format according tothe manufacturer's instructions. Comparisons of continuous variables andrisk factors that may influence variations in grade 3 or 4 CRSdevelopment were compared using the Mann-Whitney U test for 2 groups.Fisher's exact test was used to evaluate the influence of categoricalvariables on grade 3 CRS between 2 groups. Correlations were calculatedusing a rank-based Spearman test. Overall survival (OS) andleukemia-free survival (LFS) probabilities were determined by theKaplan-Meier method using all enrolled patients to determine OS andthose with MRD-negative responses for LFS. All quoted P values are twosided, and P values less than 0.05 were considered statisticallysignificant.

CD19+-RFP and Red Fluorescent Protein (RFP) were lentivirally transducedinto K562 to produce CD19-RFP-K562 cells and K562-RFP cells,respectively. The cytotoxic activity of the CD19CAR T cells was measuredbefore infusion by co-culture with the target cells, CD19-RFP-K562 cellsor K562-RFP cells, at varying ratios of effector cell to target cell(E:T). The target cells were plated into 96-well microwell plates (Nunc)at 10⁴ cells per well in 50 μl of RPMI 1640 media supplemented with 10%FBS (Gibco). The CD3/CD28 beads were removed, and effector T cells weremixed with target cells in the wells at the indicated E:T ratio. Thetotal volume was 200 μl per well. After 24 hrs of incubation, the cellswere pipetted up and down in the 96-well microwell plates with amulti-channel pipettor to dissociate the cells into single-cellsuspensions. The surviving RFP target cells in each well werephotographed, and the number of surviving RFP target cells was countedand compared with those in the wells without effector cells. The celldeath rate was calculated as (control-sample)/control×100%. Supernatantswere also collected and quantified using the human IFN-γ Valukine ELISAKit (R&D systems).

Genomic DNA was extracted using a QIAamp DNA Blood Mini Kit (Qiagen)from cryopreserved peripheral blood and bone marrow. Quantitativereal-time PCR was performed in triplicate using the ABI 2×TaqManUniversal Master Mix with AmpErase UNG (Applied Biosystems) in a 7500real-time PCR system (Applied Biosystems). Copy numbers per microgram ofgenomic DNA were calculated from a standard curve of 10-fold serialdilutions of purified CAR plasmid containing 102-108 copies/μL.Amplification of an internal control gene was used for normalization ofDNA quantities. Primers/probes specific for the CART19 transgene and aninternal control gene were as previously described (See Gokbuget N. etal., Blood 2012; 120:2032-41 and O'Brien S. et al, J Clin Oncol 2013;31:676-83).

Therapy response was assessed by flow cytometry and morphology. Whenpossible, chimeric gene expression levels were assessed in the patients.The response type was defined as MRD-negative, complete response,complete response with incomplete count recovery, stable disease andprogressive disease, as previously described. MRD-negative was definedas less than 0.01% marrow blasts by flow cytometry. Complete responsewas defined as less than 5% marrow blasts, absence of circulatingblasts, and no extramedullary sites of disease with absolute neutrophilcounts of 1000 per μL or more and platelets 100,000 per μL or more.Complete response with incomplete count recovery was defined as acomplete response with cytopenia. Stable disease was defined as diseasethat did not meet the criteria for complete response, complete responsewith incomplete count recovery, or progressive disease. Progressivedisease was defined as worse M status or no change in M status but agreater than 50% increase in absolute peripheral blast count. AfterCART19 therapy, patients were followed up every week and underwent bonemarrow examination including morphology, MRD status, chimeric geneexpression and a CART cell count every 4 weeks.

The samples were collected in gel tubes and stored at 4° C. untilcentrifugation later the same day. All blood and CSF samples were thencentrifuged at 5000 rpm for 6 minutes. The supernatants were transferredfor subsequent analysis. The BD Cytometric Bead Array Human Th1/Th2/Th17Cytokine Kit (BD Biosciences), FCAP Array v3.0 software (BDBiosciences), and a BD FACS CANTO II (BD Biosciences) were used for themeasurement and analysis of the concentrations of the cytokines such asIL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α et al., according tothe manufacturers instructions.

Erythrocyte-lysed whole BM samples were used for immunophenotyping onthe day of bone marrow aspiration. Antigen expression of blast cells wassystematically analyzed by flow cytometry (FACSCalibur flow cytometer,BD Biosciences, San Jose, Calif.) using four-color combinations ofmonoclonal antibodies (mAbs) with fluorescein isothiocyanate (FITC),phycoerythrin (PE), allophycocyanin (APC), and phycoerythrin-cyanin 7(PE-Cy7). Cell-Quest software (Becton Dickinson Biosciences) was usedfor data analysis. Monoclonal antibodies were purchased from thefollowing manufacturers: BD Biosciences, CD10-APC, CD19-FITC, CD22-PE,CD34-PE, CD45-PE-Cy7, cyCD79a-PE, surface immunoglobulin (sIg) M-PE,cytoplasmic immunoglobulin (cIg) M-APC; Beckman Coulter, CD20-APC,sIg-Lamda-FITC, sIg-Kappa-APC.

For the investigation of Minimal residual disease (MRD), the combinationof mAbs was based on the aberrant phenotypes of leukemic blasts atdiagnosis individually, and at least 500,000 events were acquired. TheMRD result was presented as the percentage of cells with aberrantphenotypes among nucleated cells. A sensitivity of 0.01% was achieved inall samples analyzed. The instrument setup was calibrated daily byanalyzing Calibrite™ beads and standard blood samples (BD™ Multi-CheckControl from BD Biosciences or CD-chex™ Plus from Streck, Inc.) forquality control.

Three patients with relapsed/refractory Chronic Lymphocytic Leukemia(r/r ALL) were treated with CD19-CAR T cells. Results are summarizedbelow in Tables 10-13. These results demonstrate that T cells expressingCD19 CAR establish antitumor effects in patients with r/r ALL. Inaddition, IL-6 and IFNγ were significantly elevated in the blood ofthree patients after transmission of CD19 CART cells compared to otherfactors (Tables 11-13). Therefore, in order to help CAR T cells achievetherapeutic effects in hematoma in the treatment of solid tumors, IL-6and IFNγ were first selected to be expressed or overexpressed inmodified T cells for treating solid tumors.

TABLE 10 CART Dosage Patient ID Tumor type (×10⁶/kg) Response CRS gradeJPDX B-ALL 4.8 MRD 3 (—) FPCY B-ALL 1.7 MRD 4 (—) SPJP B-ALL 2.8 MRD 3(—)

TABLE 11 JPDX Days IL2 IL4 IL6 IL10 TNFa IFNγ after infusion pg/mL pg/mLpg/mL pg/mL pg/mL pg/mL 1 13.56 1.11 18.62 6.58 0.96 36.55 2 3.44 1.5330.56 8.28 1.4 103.06 3 7.89 1.11 624.56 14.2 1.76 135.28 4 1.3 1.13280.87 18.28 1.97 65.3 5 7.43 3.35 211.72 30.69 3.86 66.9 6 2.64 1.5105.56 10.78 0.75 16.97 7 0 0 57.22 8.25 0 7.27 9 6.79 1.5 263.59 12.61.37 38.78 14 5.12 1.5 566.55 9.13 0 28.31 21 0 1.86 35.15 5.02 0.95 033 6.24 2.96 12.91 10.38 1.11 1.84

TABLE 12 FPCYDays IL2 IL4 IL6 IL10 TNFa IFNγ IL-17A IL-1a IL-1β GM-CSFafter infusion pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mLpg/mL 1 6.66 2.56 30.9 12.92 3.64 5.27 6.63 1.5 2.52 2.31 2 7.82 3.33 1919.7 3.61 2.29 6.37 2.32 3.12 7.29 3 2.43 0.28 13.08 22.44 0.29 1.510.86 0.19 0.55 0.4 4 0 0 10.5 26.61 0 1.1 0 0 0 0 5 3.12 1.14 23.4668.66 1.53 5.94 2.89 0.71 1.04 0.87 6 7.15 1.56 209.49 247.4 1.93 41.683.91 0.71 1.23 1.77 7 10.25 1.78 664.58 561.46 2.19 189.95 3.4 0.71 1.384.71 8 3.51 1.56 2820.04 212.49 2.03 37.74 2.89 0.61 2 1.29 9 0.8 0132.57 7.48 0 1.1 0 / 0 0 10 0 0.24 130.16 5.07 0 2.13 0 / / / 12 4.450.35 74.25 5.13 0.23 10.72 0 / / / 13 0 0 49.18 5.86 0 2.75 0 / / / 14 00 37.61 6.7 0 0 0 / / / 16 0 0.29 63.5 33.9 0 2.65 0 / / / 18 3.51 0.25725.22 266.9 0 12.24 0 0.93 1.53 0 21 3.57 0.25 424.71 76.26 0 3.15 00.93 0.64 0 30 4.33 1.7 49.76 4.86 1.76 4.28 4.25 0.59 1.37 1.05 62 4.871.7 11.21 4.86 1.76 3.34 3.7 0 1.16 0.87 90 3.23 1.2 8.7 3.83 0.1 1.882.59 0 0.29 0.87 120 0 0 6.84 7.46 0 0 0 0 0 0 128 4.34 0.39 18.42 7.990.7 1.54 1.28 0 0.47 0.15 154 11.11 2.57 29.99 11.62 6.55 2.89 5.04 0.8224.83 1.52

TABLE 13 SPJPDays IL2 IL4 IL6 IL10 TNFa IFNγ IL1a IL1β IL15 IL17A GM-CSFafter infusion pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mLpg/mL pg/mL 1 4.6 4.07 6.16 8.76 1.88 5.92 2 2.21 2.23 7.59 7.04 0.7 2.73 3.56 2.6 10.58 6.77 1.5 4.03 4 4.04 3.7 12.91 7.04 0.91 4.01 5 4.64.07 25.78 7.31 1.5 5.98 6 1.54 1.7 36.8 6.83 2.5 13.1 7 1.05 2.18 38.787.98 3.72 31.37 3.34 1.86 4.02 4.58 1.58 8 8.05 0.53 517.58 65.02 1.87228.77 1.2 5.1  0.53 7.03 2.21 9 2.97 1.26 439.82 51.75 3.67 106.21 0.691.17 0.28 2.19 1.19 10 5.15 4.45 57.42 19.86 2.27 19.26 / / / / / 141.17 0.59 38.65 4.5 0.39 5.03 / / / 0 / 20 2.22 1.78 13.48 6.42 2.7 7.97/ / / 4.35 / 30 2.13 1.87 10.72 20.22 1.24 4.54 / / / 3.12 / 60 4.121.66 8.21 5.91 1.53 2.5 0.93 1.23 / 3.4 1.12 90 3.79 1.46 25.74 7.462.01 5.72 0.06 0.96 / 3.7 1.05 123 4.87 1.94 42.87 9.47 1.76 7.7 0 0.96/ 3.7 0.87

All publications, patents and patent applications cited in thisspecification are incorporated herein by reference in their entiretiesas if each individual publication, patent or patent application werespecifically and individually indicated to be incorporated by reference.While the foregoing has been described in terms of various embodiments,the skilled artisan will appreciate that various modifications,substitutions, omissions, and changes may be made without departing fromthe spirit thereof.

1. A method of enhancing cytokine release, the method comprising:introducing a polynucleotide encoding IL-12 into a modified T cellcomprising a binding molecule binding an antigen; contacting themodified T cell with a cell comprising the antigen; and measuring levelsof cytokine release of IL-6 and IFNγ of the modified T cells, whereinthe levels of cytokine release of IL-6 and IFNγ of the modified T cellsare greater than levels of cytokine release of IL-6 and IFNγ of amodified T cell comprising the binding molecule but not thepolynucleotide.
 2. The method of claim 1, wherein the polynucleotidecomprises a promoter comprising a binding site for a transcriptionmodulator that modulates expression and/or secretion of IL-12 in themodified T cell.
 3. The method of claim 2, wherein the transcriptionmodulator comprises Hif1a and NFAT, and wherein IL-12 is expressed inresponse to activation of the modified T cell in hypoxia.
 4. The methodof claim 1, wherein the binding molecule is a Chimeric Antigen Receptor(CAR).
 5. The method of claim 4, wherein the CAR binds tMUC 1, PRLR,CLCA1, MUC12, GUCY2C, GPR35, CR1L, MUC 17, TMPRSS11B, MUC21, TMPRSS11E,CD207, SLC30A8, CFC1, SLC12A3, SSTR1, GPR27, FZD10, TSHR, SIGLEC15,SLC6A3, KISS1R, QRFPR, GPR119, CLDN6, UPK2, ADAM12, SLC45A3, ACPP,MUC21, MUC16, MS4A12, ALPP, CEA, EphA2, FAP, GPC3, IL13-Ra2, Mesothelin,PSMA, ROR1, VEGFR-II, GD2, FR-α, ErbB2, EpCAM, EGFRvIII, PSCA, or EGFR.6. The method of claim 4, wherein the CAR comprises an extracellulardomain, a transmembrane domain, and an intracellular domain, theextracellular domain binding an antigen.
 7. The method of claim 6,wherein the intracellular domain comprises a co-stimulatory domain thatcomprises an intracellular domain of a co-stimulatory moleculecomprising CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ora combination thereof.
 8. The method of claim 6, wherein the antigencomprises Epidermal growth factor receptor (EGFR), Variant III of theepidermal growth factor receptor (EGFRvIII), Human epidermal growthfactor receptor 2 (HER2), Mesothelin (MSLN), Prostate-specific membraneantigen (PSMA), Carcinoembryonic antigen (CEA), Disialoganglioside 2(GD2), Interleukin-13Ra2 (IL13Rα2), Glypican-3 (GPC3), Carbonicanhydrase IX (CAIX), L1 cell adhesion molecule (L1-CAM), Cancer antigen125 (CA125), Cluster of differentiation 133 (CD133), Fibroblastactivation protein (FAP), Cancer/testis antigen 1B (CTAG1B), Mucin 1(MUC1), Folate receptor-α (FR-α), CD19, FZD10, TSHR, PRLR, Muc 17,GUCY2C, CD207, CD3, CD5, B-Cell Maturation Antigen (BCMA), or CD4. 9.The method of claim 1, wherein the polynucleotide is in a recombinantDNA construct, in an mRNA, or in a viral vector.
 10. The method of claim1, wherein expression of the polynucleotide is regulated by SynNotchpolypeptide.
 11. The method of claim 1, wherein the binding molecule isa modified TCR.
 12. The method of claim 11, wherein the TCR is derivedfrom spontaneously occurring tumor-specific T cells in patients.
 13. Themethod of claim 11, wherein the TCR binds a tumor antigen.
 14. Themethod of claim 13, wherein the tumor antigen comprises CEA, gp100,MART-1, p53, MAGE-A3, or NY-ESO-1, or the TCR comprises TCRγ and TCRδChains or TCRα and TCRβ chains, or a combination thereof.
 15. The methodof claim 1, wherein the modified T cell further comprises an additionalCAR binding an additional antigen of a white blood cell (WBC), and theadditional antigen is different from the antigen.
 16. The method ofclaim 1, wherein the modified T cell further comprises a dominantnegative form of PD-1.
 17. The method of claim 1, wherein the modified Tcell has a reduced expression of endogenous TRAC gene.
 18. The method ofclaim 1, wherein the modified T cell is a T cell derived from a primaryhuman T cell isolated from a human donor.
 19. The method of claim 1,wherein expression of IL-12 is regulated by an inducible gene expressionsystem.
 20. The method of claim 1, wherein the binding molecule is abispecific CAR binding two different antigens.